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Extending Atmospheric Pressure Mass Spectrometry: Desorption and Ionization Considerations

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58f02a4c1c672163399f92031cbab56155a3d2d3



PAGE 1

EXTENDING ATMOSPHERIC PRE SSURE MASS SPECTROMETRY: DESORPTION AND IONIZATI ON CONSIDERATIONS By KEVIN TURNEY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Kevin Turney

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This dissertation is dedicated to my parents, Marsha and James, fo r their support; to my sister, Angela, for her frie ndship; and to my wife, Kimberly, for her patience. Strange how much you’ve got to kno w before you know how little you know. —unknown

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iv ACKNOWLEDGMENTS Any success that I have had is entirely b ecause of the people who have mentored, guided, and literally pushed me in the right directions. I could never show my full gratitude, yet below is my brief and pathetic attempt. I am indebted to my research advisor, Willard W. Harrison, for welcoming me into his laboratory and the Harrison Group family. Through the years I have had a chance to admire his scientific passion and managerial w it. The discussions in his office and at the card table will always be remembered. The greatest benefit as a Harrison group member has been the connection shared with Dr. James D. Winefordner’s Laboratory. As a research professor, Doc is supportive and compassionate of every graduate student. Also, I am grateful to Dr. Ben Smith for encouragement, and an occasional burger. It has been a delight to work alongside the Winefordner group members, enjoying the tradit ions and the history of the lab. The JDW laboratory is truly a special place. Many members of the chemistry department have been both my colleagues and my friends. Dr. Eric Oxley has continued to support the Harrison group even after his departure. I am appreciative of his friends hip and his strength in lifting motorcycles. There were times during gradua te school that only a talk with Dr. Paige Oxley could brighten my day—I still miss the discussions. I thank Li Qian for teaching me science and game strategy.

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v Some people always wear a smile, walki ng through the lab making everyone feel just a little bit better. Over the last few years, for the Winefordner and Harrison groups, that person has been Dr. Tiffany Correll. Her sense of humor a nd good-natured spirit are truly unique. She always knew how to put me in my place— Go Dumplings Lock two people in a room for a few year s and they may become friends or they may become enemies. I am happy to say the ye ars I spent with my lab mate were filled with scientific discussion, data collection, and laugher. I consider Dr. Elizabeth Pierz Hastings a cherished and respected friend. Many thanks also go to Dr. Wiehong Tan’ s laboratory, the members provided me with scientific instrumentation and camarade rie. Special thanks go to a good friend, Tim Drake, for this help with sc ience and univers ity politics. I may forget the science I learned along th e way, but I will not forget the friends I made. My most prized memories of gradua te school are the late nights and the coffee runs—both of which were not conducted alone. My path to graduate school was long and not without ordeals. I would never have gotten here without the help of an influential Professor, Dr. Suzanne Bell. She provided an engaging scientific environm ent that made me want to be a part of science. I am deeply indebted to her for both scientific and personal growth. On a more personal note, I thank my pare nts, James and Marsha DeMotta, for their never-ending support. I try my best everyda y to simply make them proud. I thank my sister, Angela, for always being there—in good ti mes and bad. Special thanks also go to my brother-in-law, Brian; and to my three wonderful nieces, Amanda, Alexis, and Abigail.

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vi To one person I am most indebted: my wife Kimberly. She may be last on paper, but she is always first in my mind. I th ank her for her support patience, and love— more Neither this dissertation nor my graduate work would have existed without her. If I’ve failed to iterate my thanks, or I left a crucial individua l or two out, please do not fault me—as you know, I need help from time to time. "Piled Higher and Deeper" by Jorge Cham www.phdcomics.com

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vii TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................xi LIST OF FIGURES..........................................................................................................xii LIST OF OBJECTS.........................................................................................................xvi ABSTRACT....................................................................................................................xvi i CHAPTER 1 MOLECULAR MASS SPECTROMETRY.................................................................1 Historical Background..................................................................................................1 Ionization Techniques...................................................................................................1 Electron Ionization................................................................................................2 Chemical Ionization...............................................................................................5 Biomolecule Analysis...................................................................................................8 Energy-Sudden Approach.....................................................................................8 Plasma Desorption.................................................................................................9 Fast Atom Bombardment....................................................................................11 Laser Desorption Ionization................................................................................13 Matrix-Assisted Laser Desorption/Ionization.....................................................14 Mechanisms..................................................................................................16 Matrix considerations...................................................................................21 Time-of-Flight Mass Spectrometry............................................................................22 Historical Perspective..........................................................................................22 Time-of-flight Theory.........................................................................................23 Kinetic energy spreads.................................................................................25 Spatial spreads..............................................................................................26 Desorption Ionization Techniques.......................................................................27 Conclusions.................................................................................................................28

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viii 2 DESIGN OF AN ATMOSPHERIC PRES SURE MATRIX-ASSISTED LASER DESORPTION/IONIZATION SO URCE FOR AN ORTHOGONALACCELERATION TIME-OF-FL IGHT MASS SPECTROMETER.........................29 Introduction.................................................................................................................29 Background.................................................................................................................29 Orthogonal-Acceleration Time-of-Flight............................................................29 Atmospheric Pressure Inlets................................................................................31 Atmospheric Pressure MALDI............................................................................33 Experimental Methods................................................................................................35 Mass Spectrometer..............................................................................................35 Electrospray Configuration.................................................................................37 Atmospheric Pressure MALDI Configuration....................................................39 Sample Preparation..............................................................................................41 Solid matrix..................................................................................................41 Liquid matrix................................................................................................42 Results and Discussion...............................................................................................42 Electrospray Evaluation.......................................................................................42 APMALDI Source...............................................................................................43 Prototype I....................................................................................................43 Prototype II...................................................................................................54 Adjusting Interface Parameters...........................................................................55 Conclusions.................................................................................................................61 3 LIQUID SUPPORTS FOR ULTRAV IOLET ATMOSPHERIC PRESSURE MATRIX-ASSISTED LASER DESORPTION/IONIZATION.................................62 Introduction.................................................................................................................62 Experimental Methods................................................................................................65 Atmospheric Pressure MALDI Source................................................................65 Solution Preparation............................................................................................65 Results and Discussion...............................................................................................66 Liquid Matrices...................................................................................................66 Chromophore Concentration...............................................................................66 Support Liquid Variations...................................................................................72 Solids versus Liquid Matrices.............................................................................76 Quantitation.........................................................................................................82 Mixture Analysis.................................................................................................84 Conclusions.................................................................................................................86 4 LASER DESORPTION CONSIDERATIONS USING LIQUID MATRICES AT ATMOSPHERIC PRESSURE...................................................................................87 Introduction.................................................................................................................87 Background.................................................................................................................89

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ix Experimental Methods................................................................................................90 Atmospheric Pressure MALDI oa-TOFMS........................................................90 Fluorescence Measurements................................................................................91 Matrix and Analytes............................................................................................91 Results and Discussion...............................................................................................92 Liquid Matrix Homogeneity................................................................................92 Laser Frequency..................................................................................................94 Fluence Dependence............................................................................................96 Particle Ejection.................................................................................................100 Plume Interactions.............................................................................................106 Conclusions...............................................................................................................107 5 FUNCTIONALIZED NANOPARTICLES FOR LIQUID APMALDI PEPTIDE ANALYSIS...............................................................................................................109 Introduction...............................................................................................................109 Experimental Methods..............................................................................................113 Materials............................................................................................................113 Nanoparticle Synthesis......................................................................................113 Silica C18 functionali zed nanoparticles.....................................................113 Magnetic aptamer nanoparticles.................................................................114 Matrix and Analyte Preparation........................................................................115 Instrumentation..................................................................................................115 Extraction Procedures........................................................................................116 Results and Discussion.............................................................................................119 Background........................................................................................................119 Nanoparticle Characterization...........................................................................119 Peptide Analysis by Liquid APMALDI with C18 Nanoparticles.....................122 Peptide Analysis by Liquid APMALDI with Aptamer Nanoparticles..............129 Conclusions...............................................................................................................132 6 SECONDARY IONIZATION OF LA SER DESORBED NEUTRALS FROM ATMOSPHERIC PRESSURE MATRIX-ASSISTED LASER DESORPTION/IONIZATION.................................................................................134 Introduction...............................................................................................................134 Experimental Method...............................................................................................136 Atmospheric Pressure MALDI oa-TOFMS......................................................136 Corona Discharge..............................................................................................136 Matrix and Analytes..........................................................................................138 Results and Discussion.............................................................................................139 Secondary Ionization of Desorbed Neutrals......................................................139 Corona Discharge..............................................................................................142 Discharge mode..........................................................................................142 Water clusters.............................................................................................143

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x Neutral Molecule Fluence Threshold................................................................145 Ion Transmission...............................................................................................149 Conclusions...............................................................................................................150 7 CONCLUDING REMARKS....................................................................................151 APPENDIX MALDI MATR ICES AND PREPARATION..........................................157 LIST OF REFERENCES.................................................................................................160 BIOGRAPHICAL SKETCH...........................................................................................172

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xi LIST OF TABLES Table Page 1-1 Common reagent gases for chem ical ionization techniques......................................7 1-2 Typical laser wavelengths, photon energi es, and pulse widths used for MALDI....17 2-1 Mass spectrometer conditions used to acquire electrospray spectra........................38 2-2 Mass spectrometer conditions used to acquire APMALDI spectra.........................40 4-1 Conditions used to acquire the emissi on spectrum of the CHCA liquid matrix....103 A-1 Common MALDI matrices....................................................................................158

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xii LIST OF FIGURES Figure Page 1-1 Diagram of an elec tron ionization source...................................................................3 1-2 Plot of ion yields versus electron energy...................................................................4 1-3 Diagram of a chemical ionization source...................................................................6 1-4 Plot of rate constants for d ecomposition and vaporization versus 1/T .....................10 1-5 Diagram of californium plas ma desorption ionization source.................................11 1-6 Diagram of a fast atom bo mbardment ionization source.........................................13 1-7 Diagram of a matrix-assisted laser desorption/ionization source.............................16 1-8 Diagram demonstrating the principle theo ry in time-of-flight mass analysis. .......24 2-1 Diagram depicting the complementary na ture of atmospheric pressure ionization sources......................................................................................................................34 2-2 Diagram of the orthogonal-acceleration time-of-flight mass spectrometer used in the studies presented is shown.................................................................................35 2-3 A photograph of the electrospray tip used for mass spectrometer characterization.38 2-4 A diagram of the components in a typi cal atmospheric pressure matrix-assisted laser desorption/ionization source............................................................................39 2-5 Plots showing the A) tota l ion count chromatogram and B) a mass spectrum for the analysis of reserpine.................................................................................................44 2-6 Plots showing the A) tota l ion count chromatogram and B) a mass spectrum for the analysis of verapamil................................................................................................45 2-7 A photograph of the first construc ted APMALDI source (Prototype I)..................47 2-8 A mass spectrum of reserpine an alyzed in a solid DHB matrix...............................48 2-9 Plot of the total ion count chroma tograms for solid and liquid matrices.................49

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xiii 2-10 A photograph of the altered target a ssembly for liquid matrix analysis..................50 2-11 Mass spectrum of reserpine in a DHB liqui d matrix taken using the modified target assembly...................................................................................................................51 2-12 A photograph of the source and target assembly with the curtain gas cover removed....................................................................................................................52 2-13 Mass spectra obtained with the target placed at a distance of A) 1.5 mm from the orifice and B) 2 mm from the orifice.......................................................................53 2-14 A photograph of the second AP MALDI source (Prototype II)................................54 2-15 Three mass spectra taken using gas flow rates of A) 0 Lmin-1, B) 1 Lmin-1, and C) 5 Lmin-1...................................................................................................................56 2-16 An illustration of the ma ss spectrometer interface...................................................57 2-17 A three dimensional plot showing the re lationship of target and nozzle voltage to analyte ion yields......................................................................................................58 2-18 A three dimensional plot showing the relationship of nozzle and skimmer voltages to analyte ion signal intensity...................................................................................59 3-1 The plot shows analyte ion and matrix background intensity as a function of CHCA concentration in the liquid matrix............................................................................68 3-2 Mass spectrum of five pi comoles of angiotensin II an alyzed using an optimized CHCA liquid matrix.................................................................................................70 3-3 An illustration of the UV-Vis absorp tion spectra collected for common MALDI matrices....................................................................................................................71 3-4 A plot of analyte ion intensity as a function of DHB concentration........................72 3-5 A chart of analyte intens ity versus the percentage of DEA in the liquid matrix......74 3-6 A chart of analyte intensity versus so lvent liquid used in the liquid matrix............75 3-7 Mass spectra of bradykinin fragment 1-7 comparing solid and liquid matrix preparations..............................................................................................................77 3-8 Mass spectra of angiotensin I compari ng solid and liquid matrix preparations.......78 3-9 Mass spectra of ACTH fragment 1839 comparing solid and liquid matrix preparations..............................................................................................................80 3-10 Mass spectra of P14R comparing solid and liquid matrix preparations....................81

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xiv 3-11 A calibration curve for angiotensin II......................................................................83 3-12 A calibration curve for bradykinin fragment 1-7.....................................................84 3-13 A mass spectrum for a peptide mixt ure using the CHCA liquid matrix..................85 4-1 A plot showing individual ion packet s resulting from liquid matrix analysis.........93 4-2 A plot of analyte ion si gnals and pulse energy as a f unction of laser frequency.....95 4-3 A plot of small molecule analyte i on yields as a function of pulse energy..............97 4-4 A plot of peptide analyte ion yi elds as a function of pulse energy..........................98 4-5 The calibration of a UV neutral dens ity filter wheel for pulse energy.....................99 4-6 Mass spectra for reserpine illustrating fragmentation produced at higher pulse energies ( i.e. 10, 90, and 180 J)..........................................................................100 4-7 Mass spectra for bradykinin fragment 17 illustrating fragmentation produced at higher pulse energies ( i.e. 10, 90, and 180 J)......................................................101 4-8 Emission spectrum of the CHCA liq uid matrix using 337 nm excitation..............102 4-9 Fluorescence image of the laser im pinging on the liquid matrix surface...............104 4-10 Fluorescence image of particles ejected from the liquid matrix............................105 4-11 Mass spectra demonstrati ng analyte signal suppression........................................107 5-1 An illustration of the cent rifugation technique used fo r nanoparticle extractions.116 5-2 A diagram illustrating the utility of th e nanoparticles with the liquid matrix........117 5-3 An illustration of the magnetic sepa ration technique used for nanoparticle extractions..............................................................................................................118 5-4 Nanoparticle characterization using fluorescence analysis....................................120 5-5 A chart of the protonated molecular ion mass spectrometry signals for three peptides...................................................................................................................123 5-6 Mass spectra collected from nanoparticle extractions............................................124 5-7 A chart showing the effect multiple washing steps has on the sodium adduct signals during mass spectrometry analysis.........................................................................125 5-8 Mass spectra collected for a 1 M a ngiotensin II solution before and after nanoparticle extraction...........................................................................................126

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xv 5-9 A chart of the protonated molecular ion mass spectrometry signals for three varied length peptides........................................................................................................128 5-10 Mass spectra for L and D vasopressin befo re and after nanopart icle extractions..131 6-1 A diagram of the laser desorption atmos pheric pressure chemical ionization source is shown..................................................................................................................137 6-2 A photograph of the LD-APCI source...................................................................138 6-3 The total ion chromatogram fo r three modes of source operation.........................140 6-4 Mass spectra for reserpine using a CHCA liquid matrix in A) APMALDI and B) LDAPCI source modes...........................................................................................141 6-5 A figure showing the transition from a corona discharge to glow discharge.........143 6-6 A mass spectrum for water clusters produ ced from the corona discharge in air....145 6-7 A plot of the analyte ion intensity and corona current as the needle voltage is adjusted (low fluence laser desorption)..................................................................146 6-8 Mass spectra of bradykinin fragment 1-7 using low fluence APMALDI and LDAPCI modes...........................................................................................................147 6-9 Mass spectra for P14R using low fluence APMALDI and LD-APCI modes.........148 6-10 Mass spectra for ACTH fragment 1839 using low fluence APMALDI and LDAPCI modes...........................................................................................................148 6-11 A plot of the analyte ion intensity and corona current as the needle voltage is adjusted (high fluence laser desorption).................................................................149

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xvi LIST OF OBJECTS Object Page 2-1 A file showing the ion source configurat ion after the target alteration for liquid matrix analysis (1.8 mb, Prototype I.exe, repeating play file)..................................48 2-2 A file showing the orientation of the prototype II source (1.8 mb, PrototypeII.exe, playable file).............................................................................................................54 4-1 A video of the laser irradiating the liquid sample surface. (1.3 mb, Liquidregeneration.mpg, 50 seconds)......................................................................94 4-2 A video of the laser irradi ating the liquid sample demons trating particle ejection at 110 J pulse energy. (1 mb, 110pulseenergy.mpg, 10 seconds)............................105 4-3 A video of the laser irradi ating the liquid sample demons trating particle ejection at 140 J pulse energy. (1 mb, 140pulseenergy.mpg, 10 seconds)............................105 4-4 A video of the laser irradi ating the liquid sample demons trating particle ejection at 180 J pulse energy. (1 mb, 180pulseenergy.mpg, 10 seconds)............................105 4-5 A video of the laser irradi ating the liquid sample demons trating particle ejection at 180 J pulse energy—magnified view. (1 mb, 110pulseenergyzoomed.mpg, 10 seconds)..................................................................................................................105 4-6 A video of the laser irradi ating the liquid sample demons trating particle ejection at 180 J pulse energy—slow motion ( non-false color) view. (5.5 mb, 180pulseenergyslowed.mpg, 50 seconds)..............................................................105 6-1 A file showing the orientation of the LD-APCI source (1.8mb, LDAPCI.exe, repeating play file).................................................................................................137

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xvii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EXTENDING ATMOSPHERIC PRE SSURE MASS SPECTROMETRY: DESORPTION AND IONIZATI ON CONSIDERATIONS By Kevin Turney December 2004 Chair: Willard W. Harrison Major Department: Chemistry Biological mass spectrometry has received we ll-deserved attention for its role in biopolymer characterization. Ma trix-assisted laser desorptio n/ionization (MALDI) is one method that allows the ionization of large intact biomolecules. For analysis, MALDI requires a suitable matrix for energy absorption and transfer to the analyte. The most widely accepted form of matrix is some form of solid that acts as an analyte host. While compatible with the low pressure environment of a typical ion source, the matrix presents a heterogeneous sample surface. Recent a dvances have allowed MALDI to be conducted at atmospheric pressure (AP), extending its fl exibility in source design and applications. This research contributes in this area by expanding upon atmospheric pressure ionization techniques and their unique applications. To further sample analysis opportunities at atmospheric pressure, a liquid matrix for UV APMALDI analysis was developed. Li quid matrices allow possible formulations focused on desorption and ionization versus v acuum stability and s ource contamination.

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xviii The liquid matrices we examined provide a self-renewing surface that eliminates sampling heterogeneity, increases sample lifetime, and provides shot-to-shot reproducibility. Ambient condition liquid samp ling also allows measurements for laser penetration depth, fluence ranges, particle ejections, and chromophore concentration, which can add to the study of MALDI mechanistic pathways. The liquid matrix offers advantages that complement current MALDI methods. Liquid sampling reduces sample preparati on, compared to solid matrices; however, during biological analysis se parations are often the rate determining step. We have explored further reductions in preparati on time for biomolecules with the use of nanoparticles. Functionalized nanoparticles provided specific ex traction, retention, and concentration of simple biopolymers. During desorption process in MALDI, a la rge population of neutral molecules is created. A secondary ionizat ion technique, such as atmospheric pressure chemical ionization, can provide reagent i ons for interaction with laser desorbed neutrals. Results show that UV laser desorbed neutrals do intera ct with atmospheric pressure reagent ions; however, the individual optimization of each pro cess is needed. Secondary ionization of the neutral molecules provides an ave nue for probing ion-molecule chemistry. The exploitation of AP interfaces with atmospheric pressure laser desorption techniques can provide needed a dvances in biolog ical analysis.

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1 CHAPTER 1 MOLECULAR MASS SPECTROMETRY “Molecules having the same mass numbers but differing in we ight by an amount determined only by the difference in binding energies of the nuclear particles can be clearly resolved…. Extension of the use of the instrument to the resolution of heavy hydrocarbons should prove fruitful.” —Alfred O. Nier 19551 Historical Background The origins of mass spectrometry are deeply rooted in the field of atomic physics. Beginning with John Dalton, and his proposed ne w atomic theory, the idea that a unique measurable property (relative atomic we ight) existed for each element, was born.2 These masses make the basis of measurement in “m ass” spectrometry. Years after the atomic theory was developed, J. J. Thompson was able to advance instrumentation and obtain the first charge-to-mass ratios, e/m for hydrogen and oxygen.3 The instrumentation directed discoveries in both experiment al atomic physics and mass sp ectrometry. This type of paradigm, instrumentation progress leading to application-driv en discoveries, has followed the mass spectrometry fi eld throughout its existence. Even today resolution and precision advances have challenged i on formation mechanistic theories.4 Ionization Techniques All mass spectrometers comprise five major components: sample inlet, ionization source, mass analyzer, ion detector, and data acquisition system. At times, regions, such as the inlet and the ionization source, can be combined (i.e. atmospheric pressure sources ( Vida infra )). However, even when regions may seem indistinguishable, each is required

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2 for mass analysis. With mass spectrometry’s ba sis in physics, it is not surprising that the beginning dealt with ionization fundamentals. Using a combination of electrostatic and magnetic fields for spatial dispersion and photographic plates for detection, physicists focused on the ion source for further advancements.5 For instance, Thompson’s initial studies using mass spectrome try involved Goldstein’s Kanalstrahlen a glow discharge ionization technique.2 While early sources provided easy access to gases, they did not provide convenient analysis of solid samples. At least not until Arthur Dempster made use of a highfrequency spark discharge to determine the isotopic analysis of platinum, palladium, gold, and iridium.2 Ions were created in the en ergetic discharge by using conducting samples as the counter electrode, but it wa s difficult to analyze semiconductors and insulators. Typical of ionizat ion techniques, advances in the field provide new avenues for analysis ( i.e. solid samples); the advancements led to additional fundamental questions. The next step in ion source deve lopment eventually shifted the focus of mass spectrometry from the physicist’s instrumentation to the chemis t’s analytical tool. While the pioneers of American mass spectrome try, Arthur Dempster, Walker Bleakney, Kenneth Bainbridge, Alfred Nier, and John Tate researched elemental composition and pure compounds, a need arose to analyze crude samples in the petroleum industry.6 For this need to be met, new ioni zation techniques were required. Electron Ionization Initially, electron ionization (EI) became the standard for hydrocarbon analysis in the petroleum industry, produci ng fragments for structural identification and molecular pattern recognition. Electron i onization is widely used in organic mass spectrometry, and is suitable for volatile and thermally stable molecules. EI is a technique that uses

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3 energetic electrons to “hit” molecules and remove an outer electron, forming a radical ion. Devised by Dempster and improved by Bl eakney and Neir, a diagram for a typical EI source is shown in Figure 1-1.7-8 filament electron beam lenses for accelerating and focusing ions ion beam to mass analyzer electron trap Ion source block repeller ion volumeU= 70 V Figure 1-1. Diagram of an el ectron ionization source. Adapted from reference 9. If an electron ( e ) transfers enough energy to a neutra l molecule (M), exceeding its ionization energy, ejection of an elec tron generates a radical cation (M+ ): 2 M eMe (1-1) Electrons are emitted from an electrically h eated filament and accelerated to 70 eV by potential gradients. As sample molecules, t ypically vapor, enter th e ionization region, the electron beam collides with the molecules, resulting in deposition of energy. Most organic molecules only require from 8 eV to 12 eV for ionization; therefore, the additional energy retained in the molecule causes fragmentation. Figure 1-2 shows how ion yields vary with electron energy. The decreased ionization efficiency at the lower potentials is due to inefficient collisions; the ionization efficiency also declines at higher potentials due to the collision efficiency. As the electron energy increases, the molecules start to become “transparent”, thereby lowering collision probabilities. Since each

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4 electron has an associated wavelength (De Br oglie wavelength), as the electron energy increases, the wavelength decreases, dimini shing the possibility for energy transfer.10 Electron ener gy ( eV ) 10410310250 10 10-210-11 10 N2He C2H2Ions produced per cm free path length and per mmHg sample pressure Figure 1-2. Plot of ion yiel ds versus electron energy. A maximum ion yield occurs near 70 eV. Adapted from reference 10. Ionization techniques must consider both the internal energy transferred and the physicochemical properties of the analyte. Some processes are energetic causing excessive fragmentation; others produce mainly molecular ions. In electron ionization, some classes of compounds have a critical energy for fragmentation that is extremely low, such that no molecular ions are pr oduced. Lowering elect ron potentials only decreases overall ionization efficiency. It quickly became clear that a new methodology would be needed for molecular ion formation.

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5 Chemical Ionization To understand electron ionization mass sp ectra, the factors affecting ion fragmentation must be considered. As advances in ion decomposition theory led chemists to recognize carbon-carbon cleavag es and rearrangement mechanisms, energetics became the common theme for explai ning ion formation, ion fragmentation, and gas-phase interactions.11 Ion formation relates to the energy required to produce an ion from a neutral molecule. Ion fragmenta tion refers to the ener gy required to break a chemical bond. Ion interactions involve the en ergies associated with collisions of ions with neutrals, ions, or surfaces. The fundame ntal studies of ion-molecule interactions enabled an understanding of gas-phase chemistry, allowing the development of fragmentation limiting ionization techniques ( e.g. chemical ionization). Chemical ionization (CI) produces ions with little excess energy using collisions of the analyte molecules with primary ions created in the source.12 Ion-molecule collisions allow for a more controlled energy transf er process, reducing fragmentation and producing intact molecular ions The reduction of fragment ation with the production of molecular ions is termed “soft” ionization. A typical CI source is shown in Figure 1-3. The CI source, shown in Figure 1-3, uses an electron ionization filament to ionize the reagent gas, which is leaked into an ev acuated chamber. The reagent ions formed interact with the neutral gas-phase analyt e yielding positive and negative ions of the sample. The ion-molecule reactions in the CI technique ( i.e. proton transfer, charge exchange, and others) depend on the prope rties of both the reagent and analyte.12 Consequently, the choice of both becomes impor tant in the analysis process. Briefly, some aspects of the analyte and r eagent population are described below.

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6 filament electron beam lenses for accelerating and focusing of ions ion beam to mass analyzer electron trap Ion source block repellerU= 200V reagent gas Figure 1-3. Diagram of a chemical ionizat ion source. Adapted from reference 9. A proton transfer reaction can occur in CI providing the proton affinity (PA) of the analyte (A) is greater than th at of the reagent ion (B): B HAAHB(1-2) A caveat does exist. If the proton tr ansfer reaction is highly exothermic, H = PA(B) – PA(A), the excess internal energy will pr omote fragmentation, preventing maximum quasi-molecular ion formation [A+H]+. Reagent ions have characteristic proton affinities listed in Table 1-1.12 An additional mode of ionization, in CI syst ems, is charge exchange. In this case, if the recombination energy (RE) of the reag ent ion (B) is greater than the ionization energy (IE) of the analyte, an exothermic reaction proceeds: B AAB (1-3) The RE of the reactant ion is defined as the exothermicity of the gas phase reaction: B eB(1-4)

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7 For a charge exchange reaction to occu r, proton transfer reactions cannot be prominent. Proton affinities for larger orga nic molecules vary from approximately 160 to 240 kcal mol-1. Table 1-1. Common reagent gases fo r chemical ionization techniques. Reagent GasReactant Ion (BH+)Proton Affinity CH4 CH5 + 131.6 kcal mol-1 CH4 C2H5 + 162.6 kcal mol-1 H2O H+(H2O)n 166.5 kcal mol-1 CH3OH H+(CH3OH)n 181.9 kcal mol-1 C3H6 C3H7 + 179.5 kcal mol-1 NH3 H+(NH3)n 204.0 kcal mol-1 *Stable reactant ions and their proton affinities are listed. Degree of solvation depends on partial pressure of reagent gas. Thermochemical data for monosolvated proton.12 Electron ionization provides the informati on for structural elucidation that is necessary in the analysis of hydrocarbons and simple organic molecules. Chemical ionization yielded complementary informa tion with molecular ion production for high proton affinity or low ionization energy molecules. These two techniques, EI and CI, allowed chemists to accept mass spectrometry as a viable analytical technique; however, they do not provide an avenue to ionize more fragile, larger molecules.

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8 Biomolecule Analysis As the biochemical and biological fields pr ogressed, the need to measure molecular weights of biopolymers became increasingly important. Instead of a more direct measurement, molecular weight was determ ined by electrophoretic chromatographic, and ultracentrifugation methods based upon the mo lecule’s conformation, Stoke’s radius, and hydrophobicity, respectively.10 EI and CI require molecule s to be in the gas-phase, so they are only amenable to volatile th ermally stable compounds. In some cases, compounds suitable for deri vatization can also meet those requirements.12 The study of proteins, carbohydrates, cell membranes, and other large biological molecules necessitated ionization techni ques capable of producing molecular ions of fragile thermolabile molecules. This need led to the eventual development of energy-sudden methods. Energy-Sudden Approach Once again fundamental ion formation studi es helped expand ionization techniques into new territory. In this case, the understanding of energy-sudden techniques was derived from the fundamentals of ion kinetics with the basis lyi ng in decomposition and desorption kinetics. Rapid heating. If desorption and subsequent ionization take place before decomposition, a limited amount of fragmentation will occur.13 This competitive notion for evaporation and decomposition yielded the idea of “rapid heating”. The rate at which energy is deposited into a sample affects th e production of neutra l gaseous molecules over fragments. If a given compound AB is heated, it is assumed that AB will be released in the gas phase and will fragme nt into A and B. The two processes, vaporization and decomposition, can be writ ten as shown in Equations 1-5 and 1-6.14

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9 vKABAB (1-5)DK A BAB (1-6) The Arrhenius equation allows the rate cons tants of the reaction to be viewed as logarithmic expressions as show n in Equations 1-7 through 1-9. exp() kfERT (1-7) lnlnvvvKFERT (1-8) lnlnDDDKFERT(1-9) For thermally labile compounds that read ily decompose, the rate constant for decomposition ( KD) is larger than for vaporization ( Kv) at low temperatures. Because the activation energy for vaporization ( Ev) is higher than for decomposition ( ED), the slope of the vaporization reaction is steeper than that of the decomposition reaction. Figure 1-4 is a plot of ln k versus 1/T for decomposition and vaporization reactions. If the relationships based upon decompositi on and vaporization hold true, then at high temperatures, where 1/T is small, vaporization is favored over decomposition. In other words, reaching the maximum temperatur e as quickly as possible provides a high degree of desorption and limits fragmentation. Plasma Desorption Desorption techniques were the energy-s udden ionization methods developed to produce molecular ions from compounds considered intractable ( i.e. nonvolatile and thermally unstable molecules). One of th e first, demonstrated by MacFarlane and Torgerson, was plasma desorp tion mass spectrometry (PDMS).15-16

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10 Decomposition reaction Lower temperatures, decomposition favored Higher temperatures, vaporization favored Vaporization EV > EDlnk 1 / T Figure 1-4. Plot of rate constants for decomposition and vaporization versus 1/T Adapted from reference 13. Desorption and ionization occurred using ener getic fission fragments from a radioactive californium source (252Cf). Californium-252 results in primarily (97%) emission of alpha particles, yet it also undergoes (3%) spont aneous fission, emitting two multiply charged fission fragments simultaneously and in near ly opposite directions. Typical decays involved 106Tc and 142Ba with energies of 104 and 79 MeV. The ionization technique works by using the pair of fission fragment s to provide high energy collisions. One fragment penetrates a thin metal foil, releas ing a burst of secondary electrons that begin the instrumentation timing sequence. The second fission fragme nt penetrates an aluminum foil holding the sample. Ions desorbed from the sample are accelerated to energies of 10 to 20 eV, pass through a drif t tube, and are detected. Alpha particle emission is discriminated against by the pr oduction of lower kinetic energy (~4 MeV) secondary electrons. Commercialized by Bi o-Ion Nordic (Uppsala, Sweden), PDMS

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11 could analyze small proteins up to ~20 kDa.17 A diagram of the source is shown in Figure 1-5. + +fission fragment detector252Cf fission source support foil with sample layer Ion acceleration grid TOF drift region Analyte detector accelerated ions fission fragments Figure 1-5. Diagram of califor nium plasma desorption ioniza tion source. Adapted from reference 18. Fast Atom Bombardment Next in the expansion of energy-sudden ionization techniques was fast atom bombardment (FAB). Developed by Barber FAB uses high energy (5 keV) neutral atoms to impart energy ont o a target, where a nonvolatile liquid matrix ( e.g. glycerol, m -nitrobenzyl alcohol) contains analyte.19 As the energetic atoms, typically argon, hit the sample surface a shockwave is induced that ejects ions and molecules.20 The development of FAB was partially to circumvent problems with electrostatic charging upon ion impact in secondary ion mass spectrometry (SIMS), which disturbed ion source potentials.21-22 SIMS uses a focused ion beam to cause secondary ions to be emitted from a sample surface. Using th e FAB technique, intact molecular or quasimolecular ions could be generated ev en in the case of highly polar compounds, which are known to be poor candidates fo r electron and chemical ionization. Additionally, the use of a liquid matrix in FAB decreased the rapid decomposition

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12 characteristic of the harsh conditions in desorption/ionization for certain samples.23-24 Using a FAB ion source, molecules nearing 10 kDa can be observed. A diagram of a FAB ionization source is shown in Figure 1-6. Ion formation theories for FAB consist of the chemical ionization model and the precursor model.25-27 The chemical ionization model assumes formation of the analyte ions in the region directly above the liquid matr ix, referred to as the selvedge region. In this space, a plasma state similar to the r eagent gas plasma in chemical ionization can exist due to the ions created from the supply of impacting primary atoms. Constituents in this region would undergo numerous reactions including the protonati on of analytes to yield quasimolecular ions ( i.e. [A+H]+). While matrix molecules are preferentially ionized for statistical reasons, they may act as the reagent ions in a system mimicking chemical ionization. The precursor model of FAB mo stly applies to ionic analytes or samples that are easily converted to ions in th e liquid matrix. The model sugge sts that ions are preformed in the matrix and are merely transferred to the gas-phase. Support comes from observations that decreasing pH increases pr otonated analyte ion yields. Additionally, relative intensities for protonated ions seem i ndependent of partial pressure of amines in the gas phase, and dependent on acidity of the matrix.28 Chemical ionization reactions would suggest just the opposite. Furthermore, incomplete desolvation of preformed ions would explain observed matr ix adducts [A+Matrix+H]+. Although the liquid matrix provides a fres h surface layer for ion production by convection and diffusion, it does have additional requirements.22,29 The matrix must: (1) absorb the primary energy, (2) solvate the anal yte, (3) have a low vapor pressure, and (4)

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13 assist in analyte formation by yielding proton donating/accepting species upon bombardment. While a matrix can be adapted as analytes require, the wrong matrix may result in complete signal suppression.22 electron ionization of FAB gas acceleration and focusing of primary ion beam neutralization of energetic ions beam of energetic atoms lenses for ion acceleration secondary ions to mass analyzer anode filament FAB gun Xegas supply effusing neutral gas target ion volume FAB probe Figure 1-6. Diagram of a fa st atom bombardment ionization source. Adapted from reference 9. Laser Desorption Ionization Before the advent of plasma desorpti on and fast atom bombardment, laser desorption/ionization (LDI) sources were us ed to analyze low-mass organic salts and light-absorbing organic molecules.30 While cases did exist for LDI to obtain mass spectra

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14 of biomolecules, the analysis of frag ile compounds over 1000 Daltons was not routine.9,31-32 This allowed FAB and 252Cf-PDMS to be far more effective in generating bimolecular mass spectra. A number of laser systems were used fo r desorption techniques, yet infrared (IR) lasers ( e.g. CO2 and Nd:YAG) saw extended use and provided mechanistic explanations of the process.33 With instantaneous pulse energies of 100 mJ, and focused beams giving fluence values of 1 MWcm-2, thermal mechanisms were predicted. As evidence, neutral and alkali species were observed to be deso rbed from the sample beyond the actual laser pulse length.31 This indicated that as the sample was heated, thermal desorption allowed ion emission. Additionally, at longer delay times, IR LD produced lower kinetic energy ions with less fragmentation.31 Since there was also no appa rent wavelength dependence, a thermal process fit the observations.33 Although time widths of the laser pulse ranged from nanoseconds to microseconds, and rapid heating seemed possible, the probl em with laser desorption was that higher temperatures could not be reached quickly e nough to obtain intact molecular ions. What was finally needed for the method to succeed was a medium that enabled the conversion of the irradiated photons to thermal ener gy without directly h eating the analyte. Matrix-Assisted Laser Desorption/Ionization A major change in mass spectrometry occurr ed with the addition of light-absorbing compounds to sample mixtures, allowing a c ontrolled desorption/ionization event. Two matrix mixtures that allowed photon absorption were originally developed: (1) ultra-fine cobalt particles, glycerol, and analyte; and (2 ) a co-crystallization of analyte with organic matrix.13,34-37 While both methods are capable of producing mass spectra of large fragile proteins ( 100 kDa), the use of the cobalt particles is considered the first ionization

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15 method that allowed the mass spectrometr y community to think about analyzing thermally labile biomolecules. This is w hy Koichi Tanaka was awarded a portion of the Nobel Prize in 2002.13 Using the absorbing particles a llowed the collection of some of the first large biomolecule mass spectra.38 However, the co-crystallization technique developed by Hillenkamp and Karas using light-absorbing organic matrices has been the more prolific method for biomolecule analysis.39 The co-crystallization method, refe rred to as matrix-assisted laser desorption/ionization (MALDI), uses a variet y of light-absorbing ma trix molecules to control uptake of laser irradi ation and cause desorption a nd ionization of the analyte.40 Unlike the wavelength independence seen in LDI, MALDI was developed from the wavelength dependence of tryptophan analysis.35 In the simple system, the amino acid acted as the absorbing molecule. Hillenkamp later reported the matrix-assisted technique of molecular ion desorption w ith the use of a more traditional nicotinic acid matrix.41 The components of a MALDI source are straig htforward. Figure 1-7 shows the general configuration for the ion source. The source comprises a laser, sample plat e (with sample), and acceleration field for transfer into the mass analyzer. While th e source is simple in construction, the underlying processes for desorption a nd ionization are le ss than trivial.42-46 The critical parameters involve a minimum of laser wavele ngth, laser fluence, matrix formulation, and sample preparation. Additionally, the mass analyzer, ion transmission, and analyte parameters must be considered. The ma ny variables to consid er during the MALDI process have made mechanistic theories di fficult to produce; th erefore, the overall mechanism for ion formation is stil l a subject of continuing research.

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16 An understanding of ionization pathways could help to maximize ion yields, control analyte charge states and fragment ation, and gain access to new classes of analytes. Knowledge of the ion formation process may also provide rational guidelines for matrix selection, something treated more as an empirical art than a scientific process. sample lens plasma laser pulse acceleration to mass analyzer Figure 1-7 Diagram of a matrix-assisted la ser desorption/ionization source. Adapted from reference 41. Mechanisms Presently, no single chemical or physic al pathway explains all positive and negative ions in the MALDI spectrum.43 Since experimental vari ables drastically affect the mass spectra outcome, several mechanistic theories have been produced to describe each effect. The mechanisms can be divided into two categories: primary and secondary ionization.43,45-46 Primary ionization refers to the gene ration of the first ions from neutral molecules—often matrix-derived species. Secondary mechanisms involve the ions not directly generated by primary pr ocesses—usually analyte ions.

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17 Major primary mechanisms include: single molecule multi-photon ionization, energy pooling, excited-state proton transfer, disproportiona tion reactions, desorption of preformed ions, thermal ionization, and spallation. Major secondary ionization mechanisms include: gas-phase proton transfer and gas-phase cationization. Primary Ionization Reactions. Since the wavelength dependence of MALDI assisted in its eventual discovery, laser wa velength plays an important role in many mechanistic theories. With the matrix requiring energy absorption, the laser wavelength must be matched with the matrix chromophore, the most common is 337 nm from a nitrogen laser; however, Nd:YAG harmonics, exim er lines, and infrared lasers have been employed. Table 1-2 shows typical photon en ergies and wavelengths for MALDI laser systems.43 Table 1-2. Typical laser wavelengths, phot on energies, and pulse widths used for MALDI.43 Laser Wavelength Photon Energy (eV)Pulse Width Nitrogen 337 nm 3.68 4 ns Nd:YAG x3 355 nm 3.49 5 ns Nd:YAG x4 266 nm 4.66 5 ns Excimer (XeCl) 302 nm 4.02 25 ns Excimer (KrF) 248 nm 5.00 25 ns Excimer (ArF) 193 nm 6.42 15 ns Er:YAG 2940 nm 0.42 85 ns CO2 10600 nm 0.12 ~1 s

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18 Even with the importance of the wavele ngth/matrix combination, MALDI spectra do not show significantly different ions with different laser wavelengths.47 However, alterations in pulse energy and sample com position are necessary to obtain similar ion populations.48 Multi-photon Ionization. Multi-photon ionization explains the radical cations formed through the wavelength dependence of MALDI.35,49-50 The reaction shown in equation 1-10 produces matrix (M) radicals, wh ich could be key intermediates for analyte ions. nhv M Me (1-10) The criticism for the mechanism lies in th e energetics needed to ionize the matrix molecules. Two photons from a nitrogen la ser yield 7.36 eV; however, the ionization potentials (IP) for common ma trix molecules are higher ( i.e. DHB at 8.05 eV).43 The typical irradiances values, 106-107 Wcm-2, make three photon ionization unlikely.51 Recent experiments suggest that clustered matrix molecules may have lower IP, yet questions still remain about the accur acy of the solid matrix measurements.43 Energy Pooling. Direct multi-photon ionization ma y not seem plausible due to energetics, but the excited states of matrix molecules are considered a viable starting point.52-53 A possibility is that two or more se parately excited matrix molecules “pool” their energy to yield one matrix radical cation.54-55 The reaction pathways for this “energy pooling” mechanism are shown in Equations 1-11 and 1-12. 2**hv M MMMMMe (1-11) ** M MAMMAe (1-12)

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19 Equations 1-11 and 1-12 could explain th e fluence dependence associated with MALDI. A critical factor in the desorptio n/ionization process is the energy density supplied to the sample (fluence, Jcm-2); however, alterations in the irradiance (Wcm-2) seems to have less of an effect on the mass spectra.56-58 Fluence versus irradiance dependence indicates that the number of photons delivered is important, not the rate at which they arrive. Excited-state proton transfer. Furthering the excited-stat e theories is the excitedstate proton transfer mechanism, wh ich helps explain protonated species.36 The pathway is shown in Equations 1-13 to 1-15. M hvM (1-13)* M AMHAH (1-14)* M MMHMH (1-15) Most matrices are not known to be goo d excited-state proton transfer agents.49,51 However, without a better knowledge rega rding the local environment in a MALDI sample, refuting the mechanism is difficult. Disproportionation. Each laser pulse in MALDI yields both positive and negative ions. To explain this observation, dispro portionation reactions have been suggested.59 The pathway is shown in equation 1-16. 2*hv M MMMMHMH (1-16) The problem with the mechanism is that positive and negative ions are not correlated in mass spectra. The fluence thres holds for each ion polarity are also different, suggesting alternate pathways.

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20 Preformed ions. Matrix solutions are typically made from organic acids; therefore, it is realistic to ex pect that preformed ions may exist in the sample. For ionic compounds, the idea that preformed ions are desorbed is reasonable, yet it is difficult to be certain whether the ions observed are trul y preformed or the result of secondary gasphase reactions discussed in the following section.60-62 Physical mechanisms. Chemical ionization mechanisms have dominated the MALDI literature, but physical mechanisms ha ve also been considered. Both thermal ionization and spallation (structural fracture of the solid) have been suggested for IR MALDI.57,63 Using infrared lasers, penetration depth is much greater, which may result in mechanical stress.64 Additionally, since IR absorp tions are weaker, the energy per volume deposited is typically too low to fully “melt” the material desorbed. Hillenkamp has proposed that spallation is an important mechanism in this case.63 Thermally induced stress that builds faster than can be dissipate d leads to a mechanical failure of the solid and ablation of material w ithout direct vaporization.64 Secondary ionization. Molecular dynamics simulations have proposed that the MALDI plume after laser deso rption is a dense cloud contai ning single molecules, ions, and clusters.66-67 Thus, an impenetrable plume of ma terial provides the opportunity for primary ions to undergo ion-molecule r eactions, necessitating secondary ionization reactions.68 Proton transfer. If primary ions are radical ca tions then proton transfer matrixmatrix reactions can readily pr oduce protonated matrix ions:49 M MMHMH (1-17)

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21 This is similar to the proposed mech anism for protonated glycerol in FAB.69 Likewise, matrix-analyte reactions can produ ce protonated analyte ions, important in MALDI spectra. Just as in chemical ioniza tion mechanisms, the presence of protonated analyte requires a proton transfer reaction. M HAMAH (1-18) Again, as in CI the reaction proceeds when G<0.12 Since proton affinities of peptides and proteins are on the order of 240 kcalmol-1, and most measurements of MALDI matrix proton affinitie s are between 183-225 kcalmol-1, the reaction should be thermodynamically favorable.70-71 It has also been noted that protonated analyte ion intensity increases for basic residue peptides indicating a chemical io nization approach to protonation.62 Additionally, varying matrices with a standard analyte al ters the internal excitation available (due to proton affin ity differences) and a ffects the degree of fragmentation. This has been referr ed to as “hot” and “cold” matrices.71-72,76 Cationization. Gas-phase cationization as a secondary ionization mechanism describes abundant cationized adducts in MALDI spectra.73-74 Ion-molecule reactions of this type have also been proposed to expl ain the pseudo-molecular ion formed by laser desorption mass spectrometry without a matrix.63 Studies have shown that salts added to MALDI samples allow the cationization of s ynthetic polymers, adding evidence to the mechanism.75 Similar to protonation reactions, cationization requires cation affinities of the analyte to exceed that of the matrix. Matrix considerations Even with numerous mechanistic studie s, matrix choice is not systematic. Difficulties in analysis stem from both ionization and co-crystallization issues.43 The

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22 appendix contains a list of some common MALDI matric es and their corresponding analyte classes. Most use pulsed UV lasers and consequently are UV absorbers. The matrices are derivatives of benzoic acid cinnamic acid, and related aromatic compounds.76 Even when the appropriate laser/matrix system is chosen, matrices need further development for specific analyte applications. In MALDI sample pr eparation, peptides and proteins are generally solubilized in 0.1% aqueous trifluoroacetic acid at a concentration of approximately 10-5 M.42 One microliter of solution is then mixed with a saturated aqueous matrix solution (around 10-3 M), which is allowed to evaporate forming crystals. Additional crystallizatio n techniques are described in the appendix. Time-of-Flight Mass Spectrometry Historical Perspective With the development of plasma desorption, fast atom bombardment, laser desorption ionization, and matrix-assisted la ser desorption/ionization, the challenge in mass spectrometry became not the production of ions but rather development of the mass analyzer. The first mass spectrographs de vised by Thompson, Aston, and Dempster utilized magnetic and electros tatic fields for ion separation.2 While these configurations are still used as today’s high resolution d ouble-focusing spectrometers, research focusing on the reduction of magnetic fields provide d the most widely used MALDI coupled mass analyzer. As magnets became a limiting factor in mass analyzer construction, primarily due to size and cost, W. E. Stephens devised an analyzer that did not require magnetic fields. In 1946, Stephens stated:77

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23 “Such a mass spectrometer should be well suited for composition control, rapid analysis, and portable use. Magnets and stabilization equipment would be eliminated.” He described the mass analyzer, the time-of-f light (TOF) mass analyz er, in his patent as:78 “…apparatus for obtaining pulses of ions segregated according to mass-velocity relationships, collecting the ions to obtain pulses of current dispersed in time and recording the result. Separation of i ons of different masses does not depend upon slit width as in the case for a conventional mass spectrometer, but depends upon only the path length, the accelerating vo ltage, pulse length and the detecting device.” Time-of-flight Theory The basic principle of the time-of-flight mass spectrometer (TOFMS) is the measurement of time as an ion travels a fixed distance. The time is related to the ion’s mass-to-charge ratio. The simplest time-of-fli ght consists of a sour ce extraction region, a drift region, and a detector. A diagra m for a TOF is given in Figure 1-8. In the source region, a voltage is applied to a backing plate that accelerates ions to a final kinetic energy (eV):31 21 2 KEmv (1-19) 22 mv eV (1-20) The mass ( m ) and velocity ( v ) of the ion are then related to the energy it obtains. Dimensional analysis can be conducted by considering units of kg for mass, m s-1 for velocity, and using 1.60 x 10-19 JeV-1. The drift region is field free, so the ions cross the region with constant velocities that are inversely proportional to the square root of their masses.31

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24 M3 + M2 + Sample Plate L (Drift Length) Drift Space Detector TOF TOF TOF 0 0 0 I I I M1 +M3 +M2 + Drift Space Detector Ion SourcePotential0 +V0+V0M1peak Grid Figure 1-8. Diagram demonstrati ng the principle theory in ti me-of-flight mass analysis. Adapted from reference 31. 1/22 eV v m (1-21) Thus, light ions travel faster a nd arrive at the detect or sooner. Ion flight times fall in the range of 10 to 200 s depending on the spectrometer arrangement. Flight time ( t ) is then related to velocity by the length of the drift tube ( D ).31 Typical units are s for time and m for tube length.

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25 1/22 m tD eV (1-22) A general derivation of the ion flight time should include the time the ions spend in the source region, yet if the region is short with respect to the dr ift tube the resulting equation is essentially th e same as equation 1-22. Relating flight time to molecule mass re quires a mass scale that follows a squareroot law. The linear equation: 1/2tamb (1-23) allows masses to be determined with as li ttle as two known masses. While constant a relates to flight tube length a nd acceleration voltages, constant b takes into account time offsets due to the laser or detector. The mass resolving power for a mass spectrum is defined as m/ m .79 In a timeof-flight analyzer this equates to a temporal resolving power as follows: 2 mt mt (1-24) where t is commonly measured as the full widt h at half maximum (FWHM). The basic resolution equation is derived from rearrangement of equation 1-22: Kinetic energy spreads Equation 1-25 is derived from TOF theory ; however, deviations in kinetic energy and spatial formation exist.80 Ions are generally formed with some initial kinetic energy, so KE = eV + U0, where U0 corresponds to the initial ki netic energy. Kinetic energy 2 22 eV mt D (1-25)

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26 spreads can account for velocity differences in the ions, detrimental to spectral resolving power.83 Resolving power enhancements can be seen by increasing accelerating voltages ( i.e. 3-30 kV), giving the ions a total energy mu ch higher than the initial energy. Timelag focusing is also used to reduce the initial kinetic energy spreads.85 Developed by Wiley and McLaren, time-lag focusing pr ovides a time delay between the ionization period and the ion-extraction puls e. This enables ions to drift within the field-free source before extraction, allowing them to distri bute according to their initial kinetic energy.31,80 The distributions are converted to spatial spre ads. One drawback for time-lag focusing is its mass-dependent nature.31 Only a narrow range can be focused for a particular value of time-delay.80,85 Further minimization of the energy distributions, sometimes inherent in the ionization technique, can be done using a reflectron.31,84,88 The simplest of reflectrons acts as an electrostatic ion mi rror, enabling the ion kinetic energy to be converted into penetration depths. Longer times in the reflec tron yield the same total time-of-flight for isobaric ions due to their increased kine tic energy and shorten drift region times. Spatial spreads Spatial distributions occur wh en ions are formed in diffe rent regions of the source, and then are accelerated through varying distan ces in the extraction field, resulting in higher drift velocities.31,85 Using a uniform accelerating field in the extraction region yields a plane, the spatial focus plane, lo cated a distance twice th at of the extraction region. This is where isobaric ions of differing velocities would be focused.85 In this arrangement, the spatial focus plane is typica lly not located at the detector, which would minimize spatial spreads. To move the focu s plane closer to the detector, a two-stage extraction region is used.31,80,81 Additional flight time spreads can also be caused by

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27 turn-around time This is the time ions having initial velocities in the opposite direction of the flight path spend in the source. Since some time spreads cannot be preven ted, and because time distributions ( t ) are maintained as the ions approach the detector, mass resolving power is limited. However, considering the resolving power equation for time-of-flight mass spectrometers, t /2 t an increase in total time, can increase resolving power with a constant time spread. This equates to using longer flight tubes for further enhancements.86 Desorption Ionization Techniques Using a gas-phase ionization technique, su ch as EI or CI, with a linear TOF-MS requires either pulsing of the ion source or ac cumulation of the continuously formed ions before the extraction pulse.31,80 Yet, with a desorpti on/ionization technique ( i.e. MALDI) the system is simplified. Since the sample is placed on a surface, spatial distributions and ion turn-around time are less significant. The ions are formed on a plate parallel to the detector. Additionally, w ith plasma desorption occurring within 10-9 s of impact, and MALDI generally using laser puls e widths of 3 to 100 ns, ionization times are shorter than the drawout pulse resulting in minimal initial temporal distributions.31 The first commercial time-of-flight mass spectrometer was produced by the Bendix Corporation (Detroit, MI).2 The system had a mass range (a t repetition rate of 10 kHz) of about 400 amu, and a mass resolving power of 200. The Bendix spectrometer was the platform that allowed the addition of multiple ionization techniques to be examined with TOF. At this early stage, success of the TOF was limited by both its mass range and mass resolving power. Combining the use of pulsed ionization techniques with boxcar recording methods together produced an extr aordinary low duty cycle. It was not until

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28 the further development of detectors, data acquisition systems, and the coupling of desorption ionization technique s did TOF mass analyzers be come standard equipment.82 Today, time-of-flight mass spectrometers can obtain resolving powers of over 10,000 with detection limits near attomole range.42,87 Conclusions Each developmental stage of an ionizati on technique or mass analyzer affords new analytical opportunities. Wh ile the recent advances in MALDI and TOF allow for the routine study of biomolecules, subtle alterations (atmos pheric pressure and orthogonal geometry) also present additional advantages. The research in this document involves the fundamental and practical study of the pro cesses in an atmospheric pressure MALDI source coupled to a TOF mass spectrometer. The focus is on liquid matrices, their interactions at atmospheric pressure, and the prospective analytical utility they provide.

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29 CHAPTER 2 DESIGN OF AN ATMOSPHERIC PRES SURE MATRIX-ASSISTED LASER DESORPTION/IONIZATION SOURCE FOR AN ORTHOGONAL-ACCELERATION TIME-OF-FLIGHT MASS SPECTROMETER Introduction “As a pulsed technique, MALDI is easily compatible with time-of-flight mass spectrometry and has been responsible (m ore than any other technique) for the renewed interest and active deve lopment of this mass analyzer.” —Robert Cotter31 A passage from Robert Cotter’s 1997 book Time-of-Flight Mass Spectrometry reflects the general scientific commun ity agreement on matrix-assisted laser desorption/ionization (MALDI) and mass spectro metry instrumentation. Therefore, it’s ironic that recent developments in time-of -flight (TOF) instrumentation have been directed towards the use of continuous ionization sources. Background Orthogonal-Acceleration Time-of-Flight Orthogonal-acceleration time-of -flight mass spectrometry (oa-TOFMS) uses ion beam deflection techniques.88 In an oa-TOFMS, mass anal ysis is done “orthogonal” to the ion source axis. Older instruments have used beam deflection techniques to narrow the ion packets pulsed into the flight tube. With orthogonal geometry, developed by O’Halloran, deflection is taken to the extr eme (90), yielding an ability to sample continuous ion sources.88-90 For years there was minimal in terest in orthogonal geometry instruments, until the configuration’s re discovery by Dawson, Guilhaus, and Dodonov.91-

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30 92 Today the configuration provides numer ous benefits to the TOF instrumentation community. Orthogonal-acceleration TOFs offer improve d mass resolving power, duty cycle, and compatibility with continuous high-energy spread ions ( e.g. electrospray ionization (ESI)). With orthogonal deflec tion a new component of velo city, which is independent of initial ion source velocity, is added to the sampled ions.88 The decoupling of mass analysis from the ion source leads to reduced time distributions for th e ions. Instead of the initial kinetic energy distributions ( due to ionization proc esses) affecting mass resolving power, orthogonal sampling discri minates against energy spreads in the ion source axis. Axial spreads are then distributed perpendicular to the pulse-out electrode of the flight tube, and do not aff ect time resolution. Provided th e detector is large enough, the ion spread in the axial direction will disperse over the detector plate of the oa-TOF. While the oa-TOF provides a duty cycle advantage, the term has been misunderstood and at times referred to as “increased sensitivity.” Duty cycle in mass spectrometry is the ratio of the time ions are extracted for mass analysis over the total time ions are produced. In a properly arra nged oa-TOF, as an ion packet is mass analyzed, a new packet is filling the pulser region of the flight tube.88 In theory this allows for a 100% duty cycle, yet it says nothing about the analyz er transmission and detector efficiency, the parameters needed to determine instrument sensitivity. With the ability to effectively sample continuous ion sources, electrospray ionization (ESI) was one of the first techniques adopted for the oa-TOFMS.93-94 ESI, originally developed by John Fenn, is a proce ss by which ions in solution are transferred to the gas-phase with limited ion fragmentation.95-96 The coupling of ESI to oa-TOFMS

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31 affords three main advantages: increased dut y cycle, improved mass accuracy, and higher m/z capability.88,97-98 While oa-TOF mass spectrometers were used for continuous ionization source mass analyzers, MALDI sources were coupled to oa-TOFs for different reasons. Unlike continuous sources, MALDI wa s compatible with the pulsed scheme of linear TOF analyzers. However, MALDI gene rates large initial kinetic energy spreads for ions, effectively reducing mass resolving power. While ion mirrors, reflectrons, and delayed extraction correct for some energy spread distributions, improvements were needed.99 Delayed extraction, for instance, must be tuned for each desired m/z range.31 Orthogonal-acceleration TOF spectromete rs decouple the energy of the desorption/ionization processes from mass an alysis, thereby increasing mass resolving power and mass accuracy.98 Atmospheric Pressure Inlets Coupling atmospheric pressure ionization (A PI) sources to a mass analyzer requires a 107 to 108 reduction in pressure, de manding precise inlet designs and efficient vacuum pumps. Since the sampling efficiency of an API source is dependent upon the number of ions and the amount of gas that can be introduced into the orifice, multi-stage pumping systems are routinely required. The gas expans ion that occurs from atmospheric pressure to vacuum, termed a supersonic jet expansion, also complicates the design of an API source, creating two major consequences: (1) io ns must be sampled with a skimmer cone, and (2) ion-solvent clusters must be prevented.33 Skimmer cones are required due to the nature of free jet expansions. The expansion of a gas from a high-pressure regi on into a low-pressure region through a small nozzle produces a supersonic jet of gas with a narrow velocity distribution and a high flux per unit area.100 The sonic jet travels at the local speed of sound in the gas.101 The

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32 expansion generates a shock wave terminati ng at the Mach disk where gas slows and diffuses. Extracting the sample through an orifice (skimmer) placed within the region before the Mach disk, termed the zone of silence, allows for maximum ion collection.101 When a mixture of gas and ions is tran sferred from atmospheric pressure to low pressure within the MS instrument, adiaba tic cooling occurs as the mixture rapidly expands in the vacuum.102 If polar neutrals ( e.g. water or solvent) are present in the mixture at that time, condensation of these neutrals on the analyte ions occurs. The size of the formed clusters may then exceed the ma ss range of the analyzer and also lower the analyte signal by distributing it over severa l ion signals. In modern MS instruments designed for atmospheric pressure ionization (API), the problem w ith clustering is of high concern. The most common methods to prevent large ion-solvent clusters from entering the mass analyzer are the addition of: (1) an axial potential gr adient, (2) a heated bath gas, or (3) a counter-current dry bath gas. A potential grad ient between the nozzle and skimmer results in a field to accelerate the ions relative to the neutral carrier gas molecules, producing energetic collis ions that fragment the clusters.101-102 A heated bath gas allows the temperature to remain a bove the condensation value during free jet expansion. Finally, a count er-current dry bath gas pr ovides cluster prevention by removing solvent molecules, averting resolva tion. A counter-current flow also limits non-ionized material from entering the syst em, making it more tolerant to “dirty” samples. While electrostatic and electroma gnetic forces play a pivotal role in ion transmission, high-pressure sources also in troduce aerodynamic forces that affect ion trajectories.

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33 Atmospheric Pressure MALDI The advent of the oa-TOF using an AP inlet allowed MALDI sources to be operated at atmospheric pressure. Laiko a nd Burlingame were the first to examine APMALDI sources for an alytical purposes.103-104 Although the oa-TOF format provided a logical means for examining the APMALDI source, additional groups coupled the new source to commercial ion trap mass spectrometers.105-107 APMALDI sources offer reduced constraint s on ion source pressure. This can be useful for high-throughput screening, wh ere automation must be used for source construction. Atmospheric pressure conditi ons also provide an opportunity to examine volatile matrices or other vacuum incompatible samples. Furthermore, an APMALDI source could be interchangeable with othe r AP sources on an atmospheric pressure interface mass spectrometer. The last benef it, interchangeability, allowed the original APMALDI investigators to fo rm a company, Mass Technolog ies, directed at producing commercial APMALDI sources for a variet y of currently employed spectrometers.108 With interchangeable ion sources a user can add utility to an already high cost instrument. Each ionization process allows for a range of chemical classes to be ionized, and in specific cases may provide compleme ntary information. Figure 2-1 shows the complementary nature of atmospheric pressure ionization sources for mass analysis. ESI may dominate the liquid chromatography ma rket due to eased liquid separations coupling, but MALDI remains the method of choice for pep tide/protein identification. By most accounts, APMALDI spectra are “similar” to vacuum MALDI spectra.105 Therefore, our initial experimental goals we re not focused on spectra evaluation, but the opportunities available for additional analytical utility using APMALD I. Furthermore,

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34 with the limited history of APMALDI, the study of desorption and ionization phenomena at atmospheric pressure may a ssist in understanding MALDI. Polarity200,000 15,000 1,000Molecular weight APCI ESIAPPIMALDI Figure 2-1. Diagram depicting the compleme ntary nature of atmospheric pressure ionization sources is shown. Each technique, atmospheric pressure photo ionization (APPI), atmospheric pres sure chemical ionization (APCI), electrospray ionization (E SI), and matrix-assisted laser desorption/ionization (MALDI), has a selective region of pola rity and molecular weight in which it is most effective. Adapted from reference 109. Fundamental research in MALDI has beco me more of a topic for scientific discussion, as one of the founders of MALDI, Franz Hillenkamp, recently wrote, “…it is time to go back and do some more basic research.”110 The beginning of these studies required the design, construction, and optim ization of an APMALDI source. This chapter describes the efforts to coupl e an APMALDI source to an oa-TOFMS.

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35 Experimental Methods Mass Spectrometer The mass spectrometer used is an orthogona l-acceleration time-of -flight (oa-TOF). Figure 2-2 is a diagram of the oa-TOF. Ion Source Pulsing region RF Quadrupole MCP Detector Nozzle Skimmer Turbomolecular Pumps Multiple Anodes Signal Conversion Electronics Flight Tube Heaviest Ions Lightest Ions Einzel Lens Figure 2-2. Diagram of the orthogonal-acceler ation time-of-flight mass spectrometer used in the studies presented is shown. The spectrometer is a prototype develope d by LECO Corporation (St. Joseph, MI, USA). Originally designed for an ESI source, the spectrometer uses a heated curtain gas for ion cluster prevention. Once the ions enter the 254 m nozzle orifice, they are sampled through a skimmer cone (2 mm inner diameter) located ~6 mm away. Voltages for the nozzle and the skimmer cone can be altered independently; the pressure in the region between the two components is ~3 Torr. This region is pumped by a two stage

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36 rotary vane roughing pump (E2M18, 4.7 Ls-1, BOC Edwards, Willmington, MA, USA), which also backs two turbomolecular pumps. Next, the ions travel into an RF-only quadrupole. The RF supply r uns at a fixed 1 MHz freque ncy with the quadrupole also having an adjustable DC offset of 0-300 V. A DC gradient of 0-50 V can also be applied. The region within the quadrupole is maintained at a pressure of ~0.1 Torr by the second stage of a hybrid turbomolecu lar drag pump (TMH261, 210 Ls-1, Pfeiffer, Germany). The primary stage of the hybrid pump is conne cted to the flight tube. Ions exit the quadrupole region through a 2 mm by 0.5 mm rectangular slit. An Einzel lens focuses and directs the ions into the f light tube pulser. A second tu rbomolecular pump is used to evacuate the Einzel region (TMH71, 70 Ls-1, Pfeiffer, Germany). The pulser then accelerates the ions up the flight tube (maintained at a pressure of 1.5 x 10-7 Torr). The flight tube is 50 cm long and cons tructed of printed circuit board.111 The tube is segmented into 39 electrodes, from pulser to detector. Potentials are applied to the electrodes through a voltage divider to form a parabolic potential field for the ions, designed to improve mass resolution. The detector used in the spectrometer is a chevron configurat ion dual microchannel plate (MCP) assembly. Electrons from the MCPs (4 cm by 8 cm, Hamamatsu, Japan) strike an array of 36 anodes. The detecti on system, termed a timeto-digital converter multi-anode detector (TDC-MAD), provides rapid temporal resolution (1 ns) and digitization rate (1 GHz) while allowing a large dynamic range from the multiple anodes.112 The signals from the anodes are fed into comparators and then a combiner board. The data from the combiner board is sent to a host board where successive spectra are summed and sent to an array board for compression and transfer to a PC via SCSI

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37 (Small Computer System Interface). The pulse r is operated at a frequency of 5 kHz, while the array board can output summed spect ra at a rate varying from 0.1 to 100 spectras-1. This allows the summation of 50 to 50,000 pulses for each output spectrum. Software, ChromoTof version 3.21 Beta ( LECO Corp., St. Joseph, MI, USA), allows spectral viewing. The mass spectrometer provides both positive and negative ion detection modes at a rated mass range of ~1 to 6000 m/z Resolving power is listed as ~2000 at m/z 600. Electrospray Configuration The ESI setup consisted of an uncoated pulled fused silica fiber with 30 m inner diameter and 360 m outer diameter (FS 360-100-30-N, New Objective, Woburn, MA, USA). Figure 2-3 is an optical image of the ESI tip taken using a Charge-Coupled Device (CCD) camera (5M, Pixera, Los Ga tos, CA, USA) mounted on a microscope (Edmund Scientific, Barrington, NJ, USA). The flow rate, 0.5 Lmin-1, was applied using a syringe pump (Pump 11, Harvard Apparatus, Holliston, MA, USA). A voltage of 3250 V was applied to a liquid junction contact by a power supply intern al to the mass spectrometer. The needle was placed ~10 mm from the orifice of the spectrometer and a curtain gas cover directed heated nitrogen gas (80 C at 800 mLmin-1) towards the ESI tip. The vo ltage on the curtain gas cover was set to 1550 V. Spectra were acquired at a rate of 4.17 spectras-1 (1200 pulsed packets). Reserpine and verapamil solutions were 2 ngL-1 in 50% aqueous methanol solutions with 1% acetic acid. All chemi cals and solvents were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification.

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38 100 m Figure 2-3. A photograph of the electros pray tip used for mass spectrometer characterization. Table 2-1 lists the potentials of the ion transfer optics and the mass analyzer when using an electrospray ionization source. Table 2-1. Mass spectrometer conditions used to acquire electrospray spectra. Nozzle 150 V Skimmer 65 V Quadrupole RF 300 V Quadrupole High 41 V Quadrupole Low 44 V Quadrupole Exit 22 V Focus -11 V Horizontal Deflect 4 V Vertical Deflect 1 V Einzel Focus -15 V Einzel Horizontal Deflect 3 V Einzel Vertical Deflect 1 V

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39 Table 2-1. Continued. Repeller 977 V Pusher 791 V Doorway 443 V Long Field Flattener 41.7 V Short Field Flattener 1 -80 V Short Field Flattener 2 -80 V Accelerator 1 228 V Accelerator 2 -240 V Accelerator 3 -423 V Accelerator 4 -672 V Accelerator 5 -883 V Flight Tube -4000 V Detector 2650 V Threshold 2020 Atmospheric Pressure MALDI Configuration A simplified diagram of an APMALD I source is shown in Figure 2-4. ND Filter Iris lens Mirror N2LaserTarget Figure 2-4. A diagram of the components in a typical atmospheric pressure matrixassisted laser desorption/i onization source is shown. While the arrangement of components varied throughout the design process, constant to the system were: (1) a 337 nm nitrogen laser (VSL-337-ND-S, Spectra-

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40 Physics, Mountain View, CA, USA), (2) UV enhanced aluminum coated positioning mirrors (Edmund Industrial Optics, Barrington, NJ, USA), (3) a vari able iris (Edmund Industrial Optics, Barrington, NJ, USA), (4) UV attenuating optics ( i.e. neutral density (ND) filters (Edmund Industrial Optics, Ba rrington, NJ, USA) or wheels (Reynard Corporation, San Clemente, CA, USA)), a nd (5) a fused silica focusing lens (Edmund Industrial Optics, Barrington, NJ, USA). Two sources were construc ted, prototype I and II. In each case, repetition rate of the la ser was monitored using an oscilloscope (TDS 210, Tektronics, Beaverton, OR, USA). Lase r power was measured using a pyroelectric detector (J4-09-030, Molectron Detector, Inc ., Santa Clara, CA, USA). Prototype I’s target was a 4 mm diameter stainless steel r od. The newer target, prototype II, is a 2 mm diameter gold coated post. Target positioning was accomplished in the first prototype by a stepper driven motorized xyz translational stage (CMA-12CCCL/ESP300; Newport, Irvine, CA, USA). The reduced size APMA LDI source, prototype II, incorporated a piezoelectric transducer driven xyz stage (8302/IPico Driver, New Focus, San Jose, CA, USA). Targets were insulated from the posi tioning devices and held at a voltage ranging from 0 to 2500 V using an internal powe r supply from the mass spectrometer. Table 2-2 lists the potentials of the ion transfer optics and the mass analyzer when using an APMALDI source. Table 2-2. Mass spectrometer conditions used to acquire APMALDI spectra. Nozzle 300 V Skimmer 75 V Quadrupole RF 300 V Quadrupole High 42 V Quadrupole Low 41.5 V

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41 Table 2-2. Continued. Quadrupole Exit 20 V Focus -30 V Horizontal Deflect 4 V Vertical Deflect 1 V Einzel Focus 0 V Einzel Horizontal Deflect 3 V Einzel Vertical Deflect 1 V Repeller 977 V Pusher 791 V Doorway 443 V Long Field Flattener 41.7 V Short Field Flattener 1 -80 V Short Field Flattener 2 -80 V Accelerator 1 228 V Accelerator 2 -240 V Accelerator 3 -423 V Accelerator 4 -672 V Accelerator 5 -883 V Flight Tube -4000 V Detector 2650 V Threshold 2020 V Sample Preparation Solid matrix The dried-droplet method was used for co-crystallization of the matrix and analyte.113 Saturated matrix solution was made by dissolving 10 mg of 2,5dihydroxybenzoic acid (DHB) (Sigma Aldrich, St. Louis, MO, USA) in 1 mL of 50% aqueous acetonitrile with 0.05% trifluoroacetic acid (TFA). Reserpine (Sigma Aldrich, St. Louis, MO, USA) was prepared as a stock solution of 1 nmoll-1 in 50% aqueous methanol with 0.1% TFA. Peptides were prepared at a concentration of 1 mgml-1 in 0.1% aqueous TFA, and diluted as necessary For crystallizati on, equal portions of matrix and analyte were mixed together and ~1.5 L of resulting solution was placed on

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42 target. The target was placed into positi on after the solution was dried at ambient conditions for 20 minutes. Liquid matrix Two liquid matrix preparations were used during the ion source optimization stage. The first matrix used 2,5-dihyroxybenzoic acid (DHB) in a glycerol-solvent solution at a 1:3 mass ratio. The glycerol-solvent soluti on consisted of 75% glyc erol, 15% water, and 10% methanol with 0.1% TFA. The sec ond matrix also used a 1:3 ratio, but of -cyano4-hydroxycinnamic acid (CHCA) (Sigma Al drich, St. Louis, MO, USA) with diethanolamine (DEA) (Sigma Aldrich, St. Louis, MO, USA). Each liquid matrix was sonicated for 10-15 minutes to ensure dissolution. For analysis, 1 l of matrix and 0.5 l of analyte were mixed on target. Results and Discussion Electrospray Evaluation Using a prototype mass spectrometer, it wa s important to evaluate instrument response independent of a new ionization source; therefore, the use of electrospray ionization (ESI) was incorporated into initial studies. ESI determined a reference point for ion transmission efficiency, mass reso lving power, and ion signal stability. Figures 2-5 and 2-6 show the total ion c ounts (TIC) and mass spectra for directly infused reserpine ( m/z 609) and verapamil ( m/z 456). For each electr ospray spectrum the resolving power is near 2500, determined by the full width half maximum definition ( m/m ). The percent relative standard deviat ion for ion counts is ~10%, and the mass accuracy, determined by external calibrat ion and 10 consecutive analysis of each compound, is between 6 to 8 ppm.

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43 By using a sample introduction rate of 0.5 Lmin-1, an extraction frequency of 5 kHz, a total summed transients of 1200, and a sample concentration of 2 ngL-1, the overall efficiency of ion detection can be calculated. The Reserpine mass spectrum shown in Figure 2-5B provides 1000 counts for the molecular ion (an injection amount of 0.008 ng during the acquisition time), yielding an efficiency of ~1 x 10-7 counts per molecule sampled. While the calculated effici ency is an important parameter, it is not solely a factor of the instrument; it also in cludes the electrospray process efficiency. ESI sensitivity, defined as the slope of the working curve, is determined by both the efficiency that molecules are converted to gas-phase ions and the efficiency that the formed ions are transferred through the mass spectrometer and detected.116 The fraction of ions analyzed then depends upon the transf er optics and the mass analyzer. Absolute efficiency of the ESI process is unknown; however, th e literature generally agrees that the limiting factor in sensitivity is ion transmission, not formation.116 Thus, as an instrumental parameter, the overall efficien cy obtained with ESI can be considered a maximum. With optimized ion transmission into the inlet, and estimate for signal intensities can be based upon ion formation efficiencies. APMALDI Source Prototype I The original configuration for the APMALDI source was designed to allow maximum adjustment for the optical component s; therefore, standa rd optical elements and hardware were used for the assembly. Figure 2-7 is a photograph of the prototype I source. Inset in the figure is a magni fied view of the target assembly.

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44 Reserpine0 200 400 600 800 1000 1200 0100200300400500600 Time (s)Intensity Reserpine0 200 400 600 800 1000 1200 500600700800900100011001200 m/zIntensity [M+H]+A B Figure 2-5. Plots showing the A) total ion count chromatogram and B) a mass spectrum for the analysis of reserpine. Inset in the figure is a ball and stick image of the molecule analyzed.

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45 Verapamil0 500 1000 1500 2000 2500 3000 3500 0100200300400500600 Time (s)Intensity Verapamil0 200 400 600 800 1000 1200 3005007009001100 m/zIntensity [M+H]+A B Figure 2-6. Plots showing the A) total ion count chromatogram and B) a mass spectrum for the analysis of verapamil. Inset in the figure is a ball and stick image of the molecule analyzed.

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46 The 337 nm nitrogen laser out puts a rectangular beam ar ea of approximately 5 mm by 7 mm with a pulse width of 4 ns. In this arrangement the laser beam is focused to a spot size on target of ~250 m by ~300 m in di ameter (elliptical shaped). The target is held at ~45 degrees from the normal of the lase r beam. The original electrospray curtain gas cover was modified by reducing a 22 mm diam eter section of the piece to 1 mm in thickness. In the electrospray mode, the origin al and modified covers produced identical results; however, the benefit for APMALDI was that the new cover allowed the target to move within 2.5 mm of the orifice. Solid matrix. When ion production is not c onsistent, source optimization is difficult. Since the oa-TOF is run asynchronous ly with the laser pulse single shot spectra (as done typically with vacuum MALDI on a linear TOF) produce drastically different ion intensities, negating any at tempt at parametric studies. At this point in ion source development, a continuous ion signal was needed. In a preliminary attempt, the laser repetition rate was increased to 10 Hz to produce a quasicontinuous ion beam, but increased laser fre quency rapidly depleted the solid sample. Figure 2-8 is a mass spectrum of solid matrix (DHB) obtained using the prototype I. While the quasi-molecular ion [M+H]+ is visible, DHB cluste rs are seen throughout the m/z range. With the laser operating at 10 Hz and the xyz motorized stage translated continuously (rate of 0.5 mm per minute), solid matrices still produced erratic ion production. Figure 2-9 shows total ion count (TIC) traces for the solid and liquid matrices. The lower trace demonstrates the i on signal reproducibility obtained with solid matrices. The drastic variations in the ion trace occur due to heterogeneity in the matrix

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47 crystals. The idea of a liquid matrix was appealing during this stage of development; however, to accommodate solution droplets, th e target angle needed to be altered. XYZ-translation stage Nitrogen Laser Focusing lens Target assembly Figure 2-7. A photograph of the first constructed APMALDI sour ce, prototype I. Inset in the figure is a magnified view of the target assembly.

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48 0 200 400 600 800 1000 1200 020040060080010001200 m/z N orma li ze d I n t ens it y[DHB-H2O+H]+[2DHB-2H2O+H]+[3DHB-3H2O+H]+[4DHB-4H2O+H]+[5DHB-5H2O+H]+[6DHB-6H2O+H]+[7DHB-7H2O+H]+[M+H]+ [M–C10H11O5+H]+ m/z 397 Figure 2-8. A mass spectrum of reserpine anal yzed in a solid DHB matrix is shown. Analysis was done using the prototype I APMALDI source. Liquid matrix. A photograph of the target as sembly constructed for liquid matrices is shown in Figure 2-10. Inse t in is a computer aided drawing (CAD) (Solidworks, Concord, MA, USA) of the targ et showing its relative position and the laser angle used for desorption. Additionally, a three dimensional depiction of the target assembly and its orientation to the orifice is shown using a executa ble output file from the CAD program. Object 2-1. A file showing the target alteration for the liquid matrix (1.8 mb, PrototypeI.exe, repe ating play file).

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49 0 10 20 30 40 50 60 70 80 90 100 0100200300400500Time (s)IntensityOH O H OOH 2 5 Dihydroxybenzoic acidSolid matrix Liquid matrix Figure 2-9. Plot of the total ion count chro matograms for solid and liquid matrices. DHB was used as the absorbing chromophor e with reserpine as the analyte. Figure 2-11 shows a liquid matrix mass sp ectrum collected using the new target holder. The spectrum is an improvement over the solid matrix analysis ( i.e. reduction of matrix clusters). While improvements in solid matrix preparations can also lower the background, the important feature of the liqui d matrix is the con tinuous ion production, as seen in Figure 2-9. In the TIC trace, as the laser is operated at 10 Hz, the liquid matrix sample stage is kept in a fixed position. Th is provided extended ion production, enabling interface optimization. Atmospheric pressure sampling. Critical in atmospheric pressure (AP) interfacing is cluster prevention; therefore, during source optimization the curtain gas cover, gas mass flow rates, and interface potenti als were examined as they related to maximum ion signal

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50 production. Additionally, moving ions at AP is difficult due to the number of collisions occurring. Unlike the reduced pressure flig ht tube, the mean free path of an ion at atmospheric pressure is on the order of nanometers. Target Liquid Matrix Gas Cover Figure 2-10. A photograph of the altered target assembly for liquid matrix analysis. Inset is a computer aided drawing of th e target showing the laser angle.

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51 0 200 400 600 800 1000 1200 020040060080010001200 m/zNormalized Intensity[2DHB-2H2O+H]+[M+H]+ [M–C10H11O5+H]+ Figure 2-11. Mass spectrum of reserpine in a DHB liquid matrix taken using the modified target assembly is shown. Mean free path can be expressed by the following equation: 2 kT p (2-1) where the mean free path ( ) is related to the collisional cross section ( ), pressure (p), Boltzman constant ( k ), and temperature (T).117 Since effective transmission is important in improving ion signals, the distance between the target and the orifice was reduced in an attempt to increase ion signals. To mini mize the target-orifice distance, the curtain gas cover was removed. Figure 2-12 is a photograph of the source and target assembly with

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52 the curtain gas cover removed. This image can be compared with Figure 2-10 where the curtain gas cover is in place. Figure 2-12. A photograph of the source and targ et assembly with the curtain gas cover removed. No ion signal differences were visible w ith the removal of th e curtain gas cover alone; however, when the target was positione d closer to the orifice the ion signal intensities increased ~30%. Figures 2-13 A and B show ma ss spectra obtained at the different target-orifice distances of A) 1.5 mm and B) 2 mm.

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53 0 250 500 750 1000 050010001500200025003000 m/z Intensity 0 250 500 750 1000 050010001500200025003000m/zIntensity2 mm 1.5 mm[M+H]+[M+H]+B A 0 250 500 750 1000 050010001500200025003000 m/z Intensity 0 250 500 750 1000 050010001500200025003000m/zIntensity2 mm 1.5 mm[M+H]+[M+H]+B A Figure 2-13. Mass spectra obtained with the ta rget placed at a distance of A) 1.5 mm from the orifice and B) 2 mm from the orifice. Moving the target closer than 1.5 mm from the nozzle resulted in high voltage arcs, and reductions in the voltages reduced ions signals to levels below that obtained at larger distances. Interestingly, a 25% decrease in target-orifice distance allowed a 30% increase in ion signals; however, this does not suggest a direct relationship.

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54 Prototype II The second iteration of the APMALDI source minimized optical component positioning and reduced the overall size. Fi gure 2-14 is a photograph of the second APMALDI prototype. Inset in the figure is a new target assembly that positioned the translational stage away from the orifice ax is. The impetus for moving the translational stage away from the orifice will be descri bed in Chapter 6. Additionally, a three dimensional depiction of the prototyp e II source is shown in Object 2-2. Object 2-2. A file showing the prototype II source (1.8 mb, PrototypeII.exe, repeating play file). XYZ-translation stage Nitrogen Laser Focusing lens Target assembly Figure 2-14. A photograph of the second APMALD I source, prototype II. Inset in the figure is a new target assembly that positioned the translational stage away from the orifice axis.

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55 The prototype II APMALDI source uses a sm aller diameter (2 mm) target with rounded edges. The edges reduced the possibil ity of arcing from the target to nozzle. The target is also coated with gold to mi nimize sample carryover. Target positioning is done using piezoelectric transducer driven moto rs. The smaller piez oelectric transducers not only diminished the source’s overall size, but also minimized the controller, which fit inside the mass spectrometer housing. An additional advantage of the modified source was safety. The target assembly completed a closed loop circuit to initiate all interface voltages. Accidental shocks were reduced. With miniaturized op tical components (New Focus, San Jose, CA, USA), the complete sour ce (sans laser) could be constructed inside an enclosure attached to the mass spectrometer—a key benefit for commercial compatibility. Adjusting Interface Parameters Initial measurements taken with the curtai n gas flow indicated that higher flows yielded larger analyte ion inte nsities and reduced background. The original device used a limited 1 Lmin-1 mass flow controller. To increase the range, a 5 Lmin-1 mass flow controller was added to the system. Figur e 2-15 shows mass spectra as the countercurrent gas flow is set at 0, 1, and 5 Lmin-1, respectively. In each case the analyte signal, angiotensin I ( m/z 1296.68), increased while the background, DEA dimer ( m/z 211), was reduced. Additiona lly, the ratio of sodium adduct [M+Na]+ to protonated molecular ion [M+H]+ was minimized with a 5 Lmin-1 gas flow. Considering the fundamentals of the cluster prevention mechanism using a counter-current gas flow, it seems reasonable that increased gas flows (counter-current) should provide analyte signal increas es through a reduction in clusters.

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56 0 300 600 900 1200 1500 0500100015002000m/zIntensity 0 300 600 900 1200 1500 0500100015002000m/zIntensity[M+H]+[M+H]+[M+H]+ [DEA+H]+[DEA+H]+[DEA+H]+[M+Na]+[M+Na]+0 L/min 1 L/min 5 L/min 0 300 600 900 1200 1500 0500100015002000m/zIntensity 0 300 600 900 1200 1500 0500100015002000m/zIntensity 0 300 600 900 1200 1500 0500100015002000m/zIntensity[M+H]+[M+H]+[M+H]+ [DEA+H]+[DEA+H]+[DEA+H]+[M+Na]+[M+Na]+0 L/min 1 L/min 5 L/min 0 300 600 900 1200 1500 0500100015002000m/zIntensityC B A Figure 2-15. Three mass spectra taken us ing gas flow rates of A) 0 Lmin-1, B) 1 Lmin-1, and C) 5 Lmin-1. Angiotensin II was used as the analyte in a CHCA liquid matrix. The original literature for APMALDI saw a different effect for gas flow, indicating concurrent flows provide d ion signal increases.104,106,114 In original APMALDI orientations an angled probe tip was used for the sample a nd a stream of nitrogen gas directed at the nozzle entraine d the ions. Only recently has literature pointed to countercurrent gas flows increasing ion signals.115 The effect of temperature on the preventi on of water clusters for supersonic jet expansion is known; thus, temper ature was empirically examined.102 A constant 100 C provided maximum ion signals without arcing. Higher temperatures provided no signal

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57 increases or background decreases, yet more frequent arcs between the target and the nozzle occurred. With an optimized target-orifice distance, target voltage was examined as it related to analyte ion yield. Although target volta ge can be varied in dependently, it does not function independently for ion transmission. Instead, it is related to nozzle voltage, the voltage applied to the orifice. Figure 2-16 shows two diagrams of the mass spectrometer interface. Target Skimmer 2 kV300 V 75 V nitrogen gas flow Target distance = 1.5 mm 35.5 mm Orifice = 254 m A B Figure 2-16. An illustration of the mass spectrome ter interface. The two images show A) the gas orifices and mass spectrometer in let, and B) the arrangement of the target, nozzle, and skimmer. Together the target and nozzle form the el ectric field that tr ansports ions from atmospheric pressure into the spectrometer. Figure 2-17 shows the interrelated functions of target voltage, nozzle voltage, and analyte (bradykinin fragment 1-7 m/z 756.4) ion yield.

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58 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 150 200 250 300 350A n a ly t e I n t e n s i t yT a r g e t V o l t a g e ( V )N o z z l e V o l t a g e ( V ) Figure 2-17. A three dimensional plot show ing the relationship of target and nozzle voltage to analyte ion yields. The skimmer was maintained at 75 V. Bradykinin fragment 1-7 ( m/z 756.4) was the analyte in a CHCA liquid matrix. Two events are demonstrated in Figure 2-17. First, increased ion yields occur at increased electrostatic fields. Th e maximum signals occur near 1100 Vmm-1 (1700 V at a distance of 1.5 mm); however, the signals ar e not maintained as the target and nozzle voltages are lowered. Second, the potential for the skim mer is 75 V; therefore, a reduction in the nozzle voltage reduces th e field between the nozzle and skimmer.

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59 0 500 1000 1500 2000 2500 100 150 200 250 300 350 400 450 60 80 100 120 140A n a l yt e I n t e n s i t yN o z z l e V o l t a g e ( V )S k i m m e r V o l t a g e ( V ) Figure 2-18. A three dimensional plot show ing the relationship of nozzle and skimmer voltages to analyte ion signal intensity. The target was maintained at 2 kV. Bradykinin fragment 1-7 ( m/z 756.4) is used as the analyte ion in a CHCA liquid matrix. Figure 2-18 was produced to investigate th e ion intensity relationship between the nozzle and skimmer. For Figure 2-18, a broader range of electric fiel ds, versus Figure 217, could be applied without significantly reduc ing the ion signals. In part, this may be due to reduced pressures betw een the nozzle and skimmer. Mass spectrometer conditions. In a supersonic beam the energy of the ions is mass dependent due the ions being at consta nt velocity. When using an oa-TOFMS, a

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60 mass dependent drift occurs in ion trajectories for the flig ht tube axis. Ions do not traverse the flight tube completely perpendi cular to the pulser; some flight tubes are offset to account for the trajectory angle. However, since each mass requires a slight change in trajectory, methods must be incorpor ated to allow for a large range of different masses to reach the detector. The methods in clude using a large de tector area to cover the trajectory range, providing deflection plat es for specific masses, and adding energy to the supersonic beam to minimize the initial energy spreads (a larger total energy relative to the initial kinetic energies). An alterna tive approach is to use a collisional focusing RF-only multipole device containing an inert gas at elevated pressures (0.01 – 1 Torr).9-10 The collisions with the gas reduce the aver age ion energy. Also, the multi-pole device focuses the ions to a beam as they approach the thermal energy of the gas.119-120 Thus, the beam leaving the ion guide has a smaller spa tial spread in the axis of the flight tube, and these properties are almost independent of the original parameters of the ion beam delivered by the source ( i.e. no memory of spatial or kinetic distributions from the ion formation processes).89 Further optimization of the mass spectrome ter conditions (pulse r voltages, Einzel lens voltages, etc.) was undert aken, but showed little eff ect. The optimization of the parameters for ESI and APMALDI (besides the interface and initia l ion transmission parameters) yielded similar results. The mass spectrometer voltages remain constant primarily due the sampling method of the jet expansion.117 While the conditions are not directly related to the jet expansion, they are indirectly related due to the use of the collisional focusing in an RF-only quadrupole.

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61 Conclusions This chapter demonstrates the initial in strument characterization, using an ESI source, and the construction of an APMA LDI source. Using an oa-TOFMS, the optimization of interface parameters ( i.e. target distance, gas flow, target voltage, nozzle voltage, and skimmer voltage) must be accomplished to maximize APMALDI ion collection and transmission. An added bene fit to the use of an RF-only quadrupole, besides collisional focusing, is the reducti on in ion source memory, easing spectrometer parameter adjustments. The APMALDI sour ce designed and implemented presents an ion source that would be easily interchang eable with a common ESI source configured mass spectrometer. The adjusted parameters and their relationship to ion yields demonstrates the necessity of optimizi ng atmospheric pressure transmission. Additionally, liquid matrices were introduced as the first analytical advantage for APMALDI. Further evidence for this is demonstrated in upcoming chapters.

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62 CHAPTER 3 LIQUID SUPPORTS FOR ULTRAVIOLET AT MOSPHERIC PRESSURE MATRIXASSISTED LASER DE SORPTION/IONIZATION Introduction Matrix-assisted laser desorption/ionization (MALDI) has seen widespread use in bioanalytical analysis.111 For analysis of large intact biomolecules, MALDI requires a suitable matrix to absorb energy and transfer it to the analyte. Many types of matrices exist, yet the most widely accepted today is so me form of solid, crys talline structure that acts as an analyte host. This solid, low volat ility matrix allows c onvenient application in the low pressure environment of a mass sp ectrometer ion source; however, the rigid lattice matrix presents a heterogeneous sample surface for successive laser pulses. As the crystal surface is ablated, analyte ion signa ls fluctuate due to the non-uniform sample surface.121 Enhanced homogeneity of the surfac e through sample preparation assists in providing more reproducible MALDI analyses.122-124 Despite the numerous sample preparation methods reported, sample heterogeneity remains an issue.121,125-127 A liquid matrix, with its self-renewing surf ace, eliminates the sample heterogeneity problem associated with solid matrices. Li quid sampling systems have found prior use in mass spectrometry to combat signal irregularities and to provide increased signal lifetime. Fast atom bombardment (FAB) and liquid secondary ion mass spectrometry (LSIMS) demonstrated the use of viscous liqui ds as an effective sample surface.128-129 The process of energy transfer differs in these two methods but the surface replen ishment principle is

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63 similar. Drawing on that success, laser deso rption methods have al so incorporated the use of liquid supports.34 A number of MALDI liquid supports have been studied, including neat liquids, particle suspensions, and chemical doping. N eat liquids provide direct absorption of the laser and limit matrix preparation time. Un fortunately, relatively few vacuum stable liquids provide adequate UV absorption.130-133 The addition of absorbing particles to a vacuum stable liquid, a particle suspension matrix, provides a low vol atility medium that absorbs UV wavelengths.134-137 These particle suspension matrices allow desorption and ionization, although the mechan ism for desorption is not typical of a solid MALDI matrix.45 The particle suspension matrix has been regarded to induce a thermal event, whereby rapid heating at the particle surface allows ther mal desorption of analytes.134,136137 Chemically doped liquid matrices may be more analogous to solid MALDI systems with their use of energy absorbing molecules.138-139 A variety of absorbing molecules have been used in binary mixtures with some success.132,138,140 Wang et al. developed vacuum stable chemically doped liquid matrices by using typical solid MALDI chromophores, 2,5-dihydroxybenzoic acid (DHB) and -cyano-4-hydroxycinnaminic acid (CHCA), in viscous li quids such as glycerol and diethanolamine (DEA).139 In some cases, a solubilizing agent was added to the mixture to ensure chromophore dissolution. Liquid matrices have provided excellent s hot-to-shot reproducibi lity and long-term analyte signal stability; however the liquid systems have been evaluated in a vacuum ion source—necessitating a low volatility medium.132,138 Liquid matrices placed in a vacuum environment have encountered problems w ith source contamination and high backing pressures (5x10-6 Torr), limiting their incorporation for routine analysis.132,137 The

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64 development of atmospheric pressure (AP) MALDI provides new oppor tunities for liquid matrices under conditions where sample volatility need not be as restricting.104-105,109 An APMALDI source provides good lim its of detection and has been suggested as a softer ionization method due to increased collisional cooling.114 With the MALDI ion source operating in an open ambient environment, v acuum stable matrices are not necessary. To expand sample analysis opportunities, wh ile providing the benefits of traditional MALDI chromophores, we have explored suitable liquid matrices for UV APMALDI.141 Glycerol-based liquid matrices, which absorb in the IR, have become common for use with IR lasers at 2.94 m and 10.6 m.48,114,142-143 However, due to cost and availability issues, IR laser systems are less commonl y used for MALDI applications. The UV nitrogen laser (337 nm)—simple to use, relati vely inexpensive, and readily available in many laboratories—has become the most widely used MALDI laser source.48,142 Our experimental goals focused on deve loping a UV-compatible liquid matrix by doping a typical MALDI chromophore, -cyano-4-hydroxycinnamic acid (CHCA), into a liquid medium. The liquid support comprises a solvent liquid for analyte solubility and a viscous component for signal lifetime. Form ulation of a UV absorbing liquid matrix for use at AP presents unique problems and advantages. As noted above, Wang et. al demonstrated similar studies for the formulation of chemically doped matrices optimized for a vacuum ion source.139 Out of the vacuum, matrices are not limited by vapor pressure or source contamination; however, desorption and ionizati on at AP must be characterized through alterations in the liquid systems. We show an example of an AP liquid matrix that provides an effective avenue for UV APMALDI analysis. In this chapter, we report on the use of suitable li quid matrices for UV APMALDI, showing ease

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65 of use, representative spectra, and promisi ng quantitation. The parameters studied include chromophore concentration, liquid supp ort variations, and quantitation capability. We believe this approach offers advantag es that complement current MALDI methods. Experimental Methods Atmospheric Pressure MALDI Source The mass spectrometer is an orthogonalacceleration TOFMS (LECO Corporation, St. Joseph, MI, USA), which is described in detail in Chapter 2. The APMALDI ion source used a 337 nm nitrogen laser (VSL337-ND-S, Spectra-Physics, Mountain View, CA, USA), focused by a fused silica lens, to i rradiate the sample on a gold coated target, 2 mm in diameter. The laser spot was ~250 m by ~300 m in elliptical diameter and yielded ~60 to 80 J per pulse. The target, onto wh ich the sample and matrix were deposited, was positioned relative to the MS orifice using a motorized xyz translational stage (8302/IPico Driver, New Focus, San Jose CA, USA). Held at 2 kV, the target was on-axis ~1.5 mm from and ~1 mm below the 254 m orifice, which was maintained at 300 V. Ions from the matrix/sample solution were transferred into the spectrometer using a counter-current gas flow interface. The nitrogen flow was set to 5 Lmin-1 and heated to 100 C. Solution Preparation The matrix was prepared by mixing CHCA (Sigma-Aldrich Corp., St. Louis, MO, USA) with the liquid support made from a solvent liquid and a viscous component. The solvent liquids used were ethanol, acetonitril e, acetone, and water (Fisher Scientific, Fair Lawn, NJ, USA). Aqueous solutions of trif luoroacetic acid (TFA) (Sigma-Aldrich Corp., St. Louis, MO, USA) were also used as test solvents for the liquid matrix. The viscous

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66 component was diethanolamine (DEA) (Sigma -Aldrich Corp., St. Louis, MO, USA). Each matrix preparation was sonicated and vorte xed to ensure dissolution. Peptides were prepared in aqueous acetonitrile with 0.1% TFA, unless otherwise stated. Sample analysis was conducted by spotting 0.5 L of matrix onto 0.5 L of analyte solution. Results and Discussion Liquid Matrices Current mass spectrometers dedicated to electrospray ionization ( e.g. oa-TOF) are directly amenable to APMALDI, yet the a dvantages of using non-vacuum compatible matrices have not been broadly investigated.109,143-144 The liquid matrix used in these studies comprises a chromophore, a suppor t liquid, and a solvent liquid. The chromophore, CHCA, absorbs and transfers energy to the analyt e in a controlled manner.37 The support liquid, DEA, is used as a viscous component for sample longevity; however, it also acts as a solubilizing agent for CHCA.136,139 The solvent liquid is used for reducing viscosity, allo wing for enhanced signal intensity and an increase in analyte solubility. Chromophore Concentration In solid sample MALDI, CHCA crystals are embedded with analyte, leaving the crystalline matrix to serve directly as the chromophore.37 In this mode various matrix-toanalyte molar ratios are possible, providing different degrees of analyte isolation and energy transfer.145 Matrix-to-analyte ratios are believed to play a role in effective analyte isolation.45 As the analyte molecules become more isolated from one another, efficient soft desorption and ionization occurs.

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67 In the liquid system, the matrix-to-anal yte ratio is not based only on CHCA amounts. The liquid matrix differs in that it contains water, ethanol and diethanolamine in large quantities to isolate the analyte without the additi on of the chromophore. Thus, analyte isolation within the support liquid can be maintained while chromophore concentration is studied. To evaluate chromophore con centration dependence, CHCA amounts doped into the liquid support were altered while monitoring analyte ion intensity. The liquid support c onsisted of a solvent liquid, 50% aqueous ethanol, and a viscous component, diethanolamine. Fi gure 3-1 shows the analyte and matrix background mass spectrometry ion counts as th e chromophore concentration is increased. The protonated analyt e, angiotensin II ( m/z 1046.5), and the matrix background, total ion count (TIC) below m/z 300, are plotted against CHCA concentrations. At concentrations below 100 mM, insufficient lase r energy is absorbed to effect desorption and measurable ionization of the analyte. As the concentration increases, both the analyte and matrix signals rapidly increase. Up to a pproximately 800 mM of CHCA, there is a direct relationshi p between analyte ion intens ity and the amount of added chromophore; however, further additions of ab sorber actually lowe r the analyte signal. As the chromophore concentration is altere d the absorption char acteristics of the matrix are changed. The CHCA allows the coupling of the laser energy to the solution, so with increasing chromophore concentration, the amount of laser energy absorbed in a finite volume on the surface of the solution also increases.

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68 0 2000 4000 6000 8000 10000 12000 0200400600800100012001400CHCA Concentration (mM)Background Intensity0 200 400 600 800 1000 1200 1400Analyte Intensity Background < 300 m/z [M+H]+ -cyano-4-hydroxycinnamic acid (CHCA) @ 337 nm –24,200 L mol-1cm-1 Figure 3-1. The plot shows an alyte ion and matrix backgroun d intensity as a function of CHCA concentration in the liquid matrix The matrix background is the total ion count for all species below m/z 300. Fifty picomoles of angiotensin II was spotted on target to monitor analyt e signal intensities. Chromophore concentration changes were done usi ng one liquid matrix support, 50% aqueous ethanol mixed with an equal part DEA. The Beer-Lambert law indicates that the laser intensity transmitted decays exponentially with distance and a constant related to absorber concentration.145 0 kbe (3-1)

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69 Where is incident laser flux, 0 is transmitted laser flux, b is the distance from the solution surface to the measurem ent of the transmitted power, and k is dependent upon concentration and molar absorptivity ( ). 0.434c k (3-2) Given a value of = 24,200 L mol-1 cm-1 for CHCA, we can estimate the penetration of the laser into the optically thick solution.139 Using the 1/e definition of laser penetration depth, we obtain a distance of ~300 nm fo r the 630 mM concentration. This concentration provided maximum analyt e ion signal and was us ed in the studies presented below. The concentration range st udied would suggest lase r penetration depths from 100 nm to 30 m. The penetration depth may provide some ra tionale for the trend observed in Figure 3-1. As the chromophore concentration rises, the effective penetr ation volume of the laser is lowered, thus reducing the number of analyte molecules affected by each laser pulse. For a laser spot size of ~300 m, an estimate for the effective sample volume can be determined (saturation effects for the ab sorbers were not considered due to the low laser powers used). Using the 630 mM CHCA concentration, the laser penetration volume element is estimated at ~1.5 x 104 m3, within which there would be 3 x 1014 water molecules, 2 x 1013 ethanol molecules, 2 x 1013 DEA molecules, and 3 x 1012 CHCA molecules. Considering 5 picomole s of angiotensin II loaded on target, the volume element of the laser would contai n ~75 attomoles of analyte, 4.5 x 107 angiotensin II molecules. Figure 3-2 shows a mass spectrum collected using the 630 mM CHCA matrix with 5 picomoles of angiotensin II loaded on targ et. The major analyte peak is the [M+H]+,

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70 with some sodium and potassium adducts also present in the spectrum. The insert, Figure 3-2A, is an expanded view of the protonated analyte peak showing a resolution of ~2500. The analyte also forms an adduct with DEA, [M+DEA+H]+, at m/z 1152.5. The matrix background shows peaks for protonated DEA (m/z 106) and the protonated DEA dimer (m/z 211). Other unidentified peaks also reside below m/z 300 at low intensity levels. 0 200 400 600 800 1000 0500100015002000250030003500400045005000m/zIntensity 10001050110011501200 [M+H]+[M+K]+[M+Na]+[M+DEA+H]+A Figure 3-2. Mass spectrum of 5 picomoles of angiotensin II analyzed using an optimized CHCA liquid matrix. The spectrum is a 5 minute summation with the laser operating at 20 Hz. The liquid matrix contained 630 mM CHCA and a 50% aqueous ethanol solution mixed with an equal part DEA. Figure 3A is a scaled view of the analyte peak and adducts. To further examine chromophore concen tration adjustments, a different chromophore was incorporated into the matrix. Studying a second chromophore may

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71 determine if the absorption required for deso rption and ionization is independent of the absorber. Each MALDI chromophore has a un ique absorption distribution as shown in Figure 3-3; however, all provide useful results when used with the proper analyte type and with the correct matrix-to-analyte ratio. 200250300350400450500 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 337nm UV absorption of Matrices 303nm 332nm 323nm 340nmHPA DHB SA CHCA IntensityWavelength (nm) Figure 3-3. An illustration of the UV-Vis absorption spectra collected for common MALDI matrices is shown. Adapted from reference 147. The MALDI chromophore 2,5-dihydroxybenzoic acid (DHB) is considered a general UV matrix useful for a variety of analyses As a comparison to CHCA results, DHB concentration studies were conducted. Figure 3-4 shows the analyte ion signals (substance P m/z 1347) versus DHB concentration. With a lower molar absorptiv ity at 337 nm (3800 L mol-1 cm-1), higher concentrations of chromophore (DHB) were required for maximum analyte signals. Further increases in the DHB concentration could not be studied due to solubility

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72 difficulties. A comparison of DHB to CHCA concentration at maximum analyte signals shows a ratio of ~6.8:1 (4300 mM/630 mM). 0 200 400 600 800 1000 1200 050010001500200025003000350040004500 Concentration( mM ) Analyte Intensity 0 200 400 600 800 1000 1200 050010001500200025003000350040004500 Concentration( mM ) Analyte Intensity2,5-Dihydroxybenzoic acid (DHB) @ 337 nm –3800 Figure 3-4. A plot of analyte ion intensity as a function of DHB concentration is shown. Fifty picomoles of substance P (Arg-P ro-Lys-Pro-Gln-Gln -Phe-Phe-Gly-LeuMet) was deposited on target. Chrom ophore concentration changes were done using one liquid matrix support, 50% aque ous acetonitrile with an equal part glycerol. This ratio is equivalent to the ratio of molar absorptivities 24,200/3,800 ~6.4:1. These results may point to an optimal value for abso rption required to yield analytical results; however, studies including other matrices must be compared. Support Liquid Variations The non-absorbing components in the liquid matrix are the solvent liquid and the viscous component.139 DEA has been used as a visc ous component to enable liquid analysis in vacuum MALDI; however, AP MALDI allows liquid matrix volatility constraints to be reduced.139 While the addition of DEA to a volatile solvent limits evaporation, enabling increased sample lifetimes, the ratio of viscous component to

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73 solvent liquid requires investigation. Usi ng the CHCA concentra tion that provided the largest analyte ion signal (~630mM), different ratios of solvent liquid to DEA were examined. The solvent liquid used was a 50% aqueous ethanol solution. As the viscous fraction, DEA, was decreased, the solvent liquid was increased to maintain a 1 L volume, with a fixed chromophore concentration. Reducing the DEA fraction limits the liquid sample lifetime, and thereby the available time for sample analysis; however, th e total viscosity of the liquid matrix also plays a role in analyte desorption. In creased viscosity represents strengthened intermolecular forces. In this case, more DEA in the mixture allows additional hydrogen bonding, thereby requiring more energy for de sorption to occur. Desorbed molecules require additional energy to overcome th e increased intermolecular forces, making desorption from the liquid phase more diffi cult. Figure 3-5 show s the analyte signal intensity as the percentage of DEA is increased in the liquid matrix. Above 50% DEA, analyte signal intensities de crease as viscosity increases, but the signal lifetime is prolonged. The typical anal ytical lifetime for a 50% DEA liquid matrix during laser desorption is 30 minutes. While an alysis can be completed in 1-5 minutes, increased lifetimes allow both source optimi zation and larger summation times for signalto-noise enhancements. The 25% and 75% so lutions provided signals for ~10 and ~60 minutes respectively. The 0% DEA mixtur e has a limited lifetime, < 1 minute, and provided low analyte signals w ith large variations. Also, without diethanolamine added to the matrix, the water and ethanol were not effective in solubilizing the CHCA, so the mixture was not homogeneous. The 100% DE A mixture was also a heterogeneous mixture due to the difficultly in mixing such a viscous solution.

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74 0 50 100 150 200 250 0%25%50%75%100%% DEA in liquid matrixAnalyte Intensity Figure 3-5. A chart of analyte intensity versus the percentage of DEA in the liquid matrix is shown. The signals represent 1 mi nute summed mass spectrometry signals. Five picomoles of angiotensin II was placed on target for analysis. From measured sample lifetimes, liquid re moval rates can be estimated. For the 50% DEA mixture, ~14 picoliters per laser pulse is estimated based on the amount of liquid spotted (1 L) and the amount remain ing after analysis (~0.5 L). Analyte removal rates can be determined using the analyte signal lifetime and the amount loaded. A 5 picomole sample lasts ~36,000 laser shot s, yielding a single pulse analyte removal rate in the attomole range. While rem oval rates are influenced by liquid matrix composition, the summation of analyte signals for longer periods allows enhancements of signal-to-noise.148 Composition of the solvent liquid is also important for the liquid matrix. To determine the effect that solvents have on analyte signal intensities, we examined

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75 common solvents. Figure 3-6 demonstrates the effect solvent variations have on analyte ion signal using a 50% DEA liquid matrix. 0 250 500 750 1000WaterEthanolEthanol/Water0.1% TFAAcetonitrileAcetoneAnalyte Intensity Figure 3-6. A chart of analyte intensity versus solvent liquid used in the liquid matrix. Each column is the solvent added to a 50% DEA mixture. The signals represent 1 minute summed mass spectro metry signals. Fifty picomoles of angiotensin II was placed on target for analysis. The CHCA concentration was kept constant at ~630 mM. The analyte signals for the solvent studied provided consistency in intensity and reproducibility. Spectral backgrounds for the solvents were also comparable. While a limited variety of solvents was tested with the liquid matrix, the results demonstrate a flexible range over which the ma trix can provide useful results. A select solvent may be necessary for specific analyt e solubility. Also, considering typical reverse phase liquid chromatography (LC) solv ents, alternative matr ix solvents could prove useful for online LC/APMALDI.

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76 Solids versus Liquid Matrices For liquid matrices to be us eful in analytical analysis they must provide analyte signals comparable to solid matrices. To examine the comparison, a study was conducted using the dried-droplet solid matrix prep aration and the optimized liquid matrix preparation.37 For the solid matrix analysis 1 mL of 50% ACN in 0.05% TFA solution was mixed with 10 mg of CHCA matrix. Next, 10 L of an alyte sample was mixed with an equal volume of matrix solution. One mi croliter of the mixture was spotted onto the target and allowed to dry for 20 mi nutes. Bradykinin fragement 1-7 (m/z 757.4), human angiotensin II (m/z 1046.5), human Adrenocorticotro pic (ACTH) fragment 18-39 (m/z 2465.2), and synthetic peptide P14R (m/z 1533.8) were dissolved in aqueous 0.1% TFA (Sigma-Aldrich Corp., St. Louis, MO, USA). The same peptide solutions were used for liquid matrix analysis. The liquid matrix consisted of 630 mM concentration of CHCA with a support liquid of 50% DEA and 50% aqueous ethanol. The deposited liquid matrix consisted of 0.5 L of matrix soluti on placed on top of 0.5 L of analyte solution to ensure mixing. For both solid and liquid analysis, total peptide placed on target was 25 picomoles. Figures 3-7 to 3-10 show MALDI mass spect ra for peptide analytes using either solid and liquid matrix preparations. All mass spectrometer and source conditions were kept constant. Figures 3-7 and 3-8 show bradykinin fragment 1-7and angiotensin II analyzed with both solid and liquid matrices.

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77 0 500 1000 1500 2000 2500 3000Intensity 0 500 1000 1500 2000 2500 3000 5006007008009001000m/zIntensityBradykinin1-7[M+H]+[M+H]+[M+Na]+[M+K]+[M+DEA+H]+Liquid Solid Figure 3-7. Mass spectra of bradykinin fragme nt 1-7 comparing solid and liquid matrix preparations. Twenty-five picomoles of analyte was deposited on target, and each spectrum was the summation of 100 individual spectra.

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78 0 500 1000 1500 2000Intensity 0 500 1000 1500 2000 500750100012501500m/zIntensityAngiotensinII[M+H]+[M+H]+[M+Na]+[M+K]+Liquid Solid Figure 3-8. Mass spectra of angiotensin I comp aring solid and liquid matrix preparations. Twenty-five picomoles of analyte was deposited on target, and each spectrum was the summation of 10 0 individual spectra.

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79 In both Figures 3-7 and 3-8, the protonate d molecule signals can be seen in the solid and liquid matrix formulations. The li quid matrix differs in signal intensity and adduct formation. Though the liqui d matrix presents larger [M+H]+ signals, no fluence adjustments were made to optimize solid matrix analysis; therefore, conclusions cannot be drawn on the limits of detection of liquid ve rsus solid matrices. However, what can be considered in Figures 3-7 and 3-8 is the a ppearance of alkali metal and DEA adducts. While DEA is not available in the solid matrix formulation, sodium and potassium are present. The formation of adduc ts is a known phenomenon for APMALDI.114 Adducts in the liquid matrix suggest a softer desorpti on/ionization event. Co llisional cooling at AP is thought to provide the stabilization for adducts to remain intact. Softer ionization modes can provide benefits when examini ng non-covalent interac tions or analyzing fragile compounds (e.g., deoxyribonucleic acids). Additiona lly, instrumental and sample preparation methods are availabl e to reduce analyte adducts (e.g., ultra-clean targets, high-grade reagents, larger declusteri ng voltages between noz zle and skimmer). Figure 3-9 shows ACTH fragment 18-39 analyzed with both solid and liquid matrices. In Figure 3-9 no analyte signal diffe rences were present between the solid and liquid matrix preparations. Also, CHCA cluster formation is seen with both the solid and liquid matrices. Figure 3-10 shows P14R analyzed with both solid and liquid matrices.

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80 0 250 500 750 1000Intensity 0 250 500 750 1000 200022002400260028003000m/zIntensityACTH 18-39[M+H]+[M+H]+[M+CHCA+H]+[M+2CHCA+H]+[M+CHCA+H]+[M+2CHCA+H]+Liquid Solid Figure 3-9. Mass spectra of ACTH fragment 18-39 comparing solid and liquid matrix preparations. Twenty-five picomoles of analyte was deposited on target, and each spectrum was the summation of 100 individual spectra.

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81 0 500 1000 1500 2000Intensity 0 500 1000 1500 2000 110013001500170019002100m/zIntensityP14R[M+H]+[M+H]+[M+Na]+[M+DEA+H]+y14y12y11y13y14y13Liquid Solid Figure 3-10. Mass spectra of P14R comparing solid and liquid matrix preparations. Twenty-five picomoles of analyte was deposited on target, and each spectrum was the summation of 10 0 individual spectra.

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82 For the synthetic peptide, fragmenta tion is known to occur under mild conditions.149 Both matrix formulations show y se ries ions. This ion series is the cterminal ion series formed during fragmenta tion of the peptide, so it contains the cterminal and extensions from this residue (i.e., y14 represents PPPPPPPPPPPPPR). While different matrix chromophores have show n more or less fragmentation, it appears that the liquid formulation has fewer frag ments present, again indicating a softer mechanism for desorption and ionization.150 Quantitation The liquid matrix acts as a homogeneous sampling environment for desorption and ionization, thus offering oppor tunity for quantitative analysis. To evaluate the reproducibility of liquid sampling, intra a nd inter-sampling precision was determined. With a liquid matrix sample lasting tens of minutes, ten 1 minute summed spectra could be compared. Inter-sampling precision was cal culated to be ~10-13% RSD, with intrasampling precision at ~5-7% RSD. Quantitation capabilities, without an in ternal standard, were examined by producing calibration curves for angiotensin II and bradykinin fragment 1-7. Figure 3-11 shows the calibration curve obtained for angiot ensin II using a liquid matrix. The curve was obtained by analyzing serial dilutions of a standard peptide mixture, with the volume of analyte placed on target maintained at 0.5 L. For Figure 3-11, each point represents a one minute sum with standard deviations obtai ned from five analyses. The inset shows a scaled section of the curve for the femtom ole to low picomole range. While the R2 values demonstrate the ability for direct quantitation, the dynam ic range for MALDI becomes apparent when viewing both ranges in Figure 3-11. For this reason, two analytical functions are presented, one for each range.

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83 R2= 0.995 0 1000 2000 3000 4000 5000 6000 7000 050100150200250300Picomoleson TargetAnalyte Intensity R2= 0.999 0 50 100 150 200 250 300 01234567 Figure 3-11. A calibration curve for angioten sin II. The calibration curves obtained include calculated R2 values for each analyte range. The inset shows a scaled view of the full range. Figure 3-12 shows a similar cal ibration curve for bradykinin fragment 1-7. For the bradykinin fragment 1-7 calibration curve, a smaller range was examined using reduced amounts of peptide placed on target. The current open design of the ion source may prevent further reductions in the limit of detection (LOD) values. An en closed source minimizes water vapor contributions, assists cluster prevention, and increases analyte signals. Our source uses a nitrogen counter-current gas for cluster preventio n. Typically used with ESI, the use of a nitrogen current gas with APMALDI has been more limited. Recently, Miller and Perkins described a counter-current gas (5 Lmin-1 of nitrogen gas at a temperature of 300C) for an enclosed APMALDI ion source coupled to an ion trap mass spectrometer that yielded detection limits of ~125 attomoles.115

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84 R 2 = 0.9990 2000 4000 6000 8000 10000 12000 05101520253035404550 R2= 0.935 0 100 200 300 400 500 00.20.40.60.81 Picomoles on TargetAnalyte IntensityBradykinin Fragment 1-7 Figure 3-12. A calibration curve for bradykini n fragment 1-7. The calibration curves obtained include calculated R2 values for each analyte range. The inset shows a scaled view of the full range. Declustering was determined to be the largest contributor for detection limit enhancements. Thus, by enclosing the s ource in a nitrogen purged environment, additional declustering could increase analyte signals. The detection limits shown for both calibration curves are not to the levels possible using vacuum MALDI. Ion collection and tr ansmission is most likely the limiting factor, not the liquid matrices. All values were obtained on a laboratory built ion source. Decreased LODs may be possible with in creased summation times, further source optimization, and an appropriate enclosure (to control the ion transfer environment). Mixture Analysis To demonstrate the liquid matrix’s utili ty for peptide analysis, a mixture of standard peptides was examined as a test sample. Figure 3-13 depicts a one minute

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85 summed mass spectrum for a mixture of bradyki nin fragment 1-7, angiotensin II, ACTH fragment 18-39, and insulin oxidized B chain (2 picomoles each). 0 20 40 60 80 100 700120017002200270032003700m/zIntensityBradykinin 1-7 Angiotensin II ACTH 18-39 Insulin oxidized B chain Figure 3-13. A mass spectrum for a peptide mi xture using the CHCA liquid matrix. The mixture contained angiotensin II, bra dykinin fragment 1-7, ACTH fragment 19-39, and insulin oxidized B chain. Two picomoles of each peptide were mixed and placed on target. The decay of signal intensity with in creasing mass shown in Figure 3-13 is reproducible, but somewhat puzzling. There is some indication that the input quadrupole may be one of the factors affecting this re sponse. The mass range examined was based on the available instrumentation and is not believed to be a liquid matrix limitation. An alternate RF power supply as well as pre ssure variations in the quadrupole region may assist in increasing the mass range.

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86 Conclusions The liquid matrix provides a homoge nous sample allowing easy sample preparation, increased sample lifetime, and added utility for an atmospheric pressure matrix-assisted laser desorption/ionization s ource. With the use of a typical MALDI chromophore, -cyano-4-hydroxycinnaminic acid, the matrix provides the known properties of an organic absorbing matrix w ith the added benefits of a liquid sampling system. Chromophore concentration adjustments provided insight into the necessary absorbance for UV APMALDI and demonstrat ed the importance of laser penetration depth in MALDI liquid sampling. Liquid s upport variations allowed adjustments of sample lifetime and analyte solvents. The sample lifetime is beneficial for instrument tuning and source optimization; however, t oo high a liquid viscosity lowers signal intensity. Quantitation without the use of internal standards may be possible with a liquid matrix. Reproducibility suggests that the liqui d matrix can alleviate some inconsistencies seen with solid sample MALDI. The liquid sy stem also provides a convenient avenue for fundamental studies of desorption. The m easurements for laser penetration depth, solution viscosity, and solvent additives could add to the information on MALDI mechanisms. While the liquid matrix pr ovides immediate benefits for APMALDI analysis with its ease of use, additio nal possibilities include an on-line liquid UV APMALDI ion source for chromatography or reaction monitoring.

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87 CHAPTER 4 LASER DESORPTION CONSIDERATIONS USING LIQUID MATRICES AT ATMOSPHERIC PRESSURE Introduction The production of analyte ion sign als using matrix-assisted laser desorption/ionization (MALDI) can be affect ed by both sample preparation and matrix formulation.151 Detection of the ions is determ ined by interface and mass spectrometer settings—allowing for increased ion transmi ssion. While instrument and sample/matrix parameters provide some insight into MALD I processes, the underlying mechanisms are still elusive.43 Parametric studies present the standard method for adding mechanistic information. For example, investigating the laser-sample interactions provides detailed information on photon absorption characteristic s and yields an experimental basis for future applications. Most studies are concer ned with the relationships between ion yield and laser fluence.152-153 Ejected particles have also been a focus in deciphering how the desorption process affects ion formation.154 Traditional laser modifications have incl uded alterations in absorption wavelength, pulse duration, and input fluence. Studies on wavelength dependence have shown that the matrix absorption directly relates to the wavelength used.45 While cases exist for using laser wavelengths outside of the matr ices’ major absorption bands, most studies reveal absorption-excitation dependence.155

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88 Short pulse width lasers (e.g., UV ~4 ns and IR ~6-200 ns) provide ion generation without thermal degradation of the analyte. Longer irradiation causes heating of the bulk material and extensive fragmentation.156 However, IR systems have shown that decreased pulse widths can reduce phot on flux thresholds for ion detection.157 Fluence is defined as energy input per unit area; in MALDI typical fluences are in the range of 100-1000 Jm-2.45 Irradiance is fluence divided by the laser pulse duration; MALDI irradiances are in the range of 106-107 Wcm-2.45 While MALDI shows some irradiance dependence, it is the fluence values that have direct relati onships to analyte ion yields. When fluence is below a threshold level no ion production is observed. These threshold values are a key factor fo r understanding underlying mechanisms.44 Fluence values can be used to estimate the number of excited matrix molecules, which are necessary for further ion-molecule reactions Fluence values depend on the matrix and analyte in the sample. However, the values can also vary with source pressure. Higher pressures have been noted to require higher fluence values.43,157 A recent study using APMALDI provided fluence values through th e focusing and defocusing of the laser beam onto the target. In this mode, spot size adjustments were made versus pulse energy.157 Under vacuum conditions, spot size va riations (with the corresponding pulse energy changes, keeping fluence constant) have drastically altered the measured fluence ranges.57 The variations are considered to be du e to the inconsistencies in reporting laser spot sizes.45 Large-scale molecular dynamics (MD) si mulation studies have provided valuable data to close the gap between the experiment al results and theoretical considerations.45

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89 The MD simulations show how laser ablation from organic solids ca n be characterized into two mechanisms for molecular ejection defined by fluence threshold values. Below the threshold, mainly singly charged molecule s are desorbed from the surface. Above the threshold, the ablation processes become im portant and larger mo lecular clusters are introduced into the MALDI.57,152,158 The measurements of fluence dependent ion yields may focus on the interactions of th e two theoretically proposed pathways. The studies presented in this chapter fo cus on the laser-sample interactions at atmospheric pressure using a volatile liquid ma trix. By using a UV la ser, variations in frequency and pulse energy can be related to ion yields. The homogeneity of the sample was also examined through single laser shot measurements. Pulse energy variations allowed a detailed examination of flue nce dependence on analyte ion yields. Additionally, the examination of particle ejections from the matrix at higher fluences helped explain ion signal reductions. Th ese measurements may prove useful for determining mechanistic pathways. Background While the ionization proce sses involved in MALDI are complex and still largely undetermined, much more is understood a bout the desorption phase. In MALDI desorption, a plume is formed comparable to a pulsed jet expansion. Theoretical simulations describe the UV MALDI plume as an explosive solid-to-gas phase transition. Ion emission is observed within nanoseconds of the laser pulse, wh ile neutral desorption can occur microseconds after.158 Matrix ion velocities in the plume of a solid matrix MALDI analysis can reach up to 1000 ms-1, depending upon matrix preparation and composition. Analyte ions are generally 100-200 ms-1 slower.159

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90 The mechanistic view of the MALDI plume is that primary ions are created in a hot, dense bath of neutral matrix molecule s and clusters, and unde rgo collisions before being extracted into the mass spectrometer. Thus, secondary ion-molecule reactions are expected.46 The final ions observed in MALDI mass spectra can be a result of the primary ions formed; therefore, a major goal of matrix design must be to improve the number of primary ions created while limiti ng the fraction of larg e clusters and chunks.46 During desorption, single molecules, clusters, and aggregates of matrix are ejected. The fraction of free molecules and small cluste rs is considered greatest at low fluence, yet the absolute ion yield is greatest at higher fluence.43,45 Optimizing laser fluence may minimize the amount of large clusters pr oduced and maximize the ion production. Experimental Methods Atmospheric Pressure MALDI oa-TOFMS The orthogonal-acceleration time-of-flight ma ss spectrometer (oa-TOF) used in this research was previously described in Chapte r 2. Briefly, the APMALDI source used a 337 nm nitrogen laser (VSL-337-ND-S, Spectra -Physics, Mountain View, CA, USA) for desorption pulse width ~4 ns. The la ser was operated asynchronous with the TOF acquisition pulse at 5 kHz. Pulse energies ranged from ~5-180 J, measured directly using a pyroelectric detector (J4-09-030, Mo lectron Detector, Inc., Santa Clara, CA, USA). Variations in the energy transmitted to the sample came from a gradient UV attenuating wheel (Reynard Corporation, San Clemente, CA, USA). Laser repetition rates were monitored using an oscilloscope (TDS 210, Tektronics, Beaverton, OR, USA). Target positioning was accomplished usi ng a piezoelectric transducer driven xyz stage (8302/IPico Driver, New Focus, San Jo se, CA, USA). The at mospheric pressure

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91 interface used a heated count er-current nitrogen gas (100C, 5 Lmin-1) for cluster prevention. The ion transport field be tween the target and nozzle was 1100 Vmm-1. Gas pressures in the skimmer and RF-only quadrupole were 3 Torr and 10-1 Torr respectively. Video monitoring of the matrix deposited on the target was done using a chargedcoupled device (CCD) (Sensicam, IPentamax III, Roper Scientific, Trenton, NJ, USA) fitted with an adjustable lens. Images were acquired using the manufacturer’s software (Tillvision, Roper Scientific, Trenton, NJ, USA). The spectra shown are an accumulation of summed spectra for 1-5 minutes. Fluorescence Measurements The emission profile for CHCA in soluti on was taken using a plate reader (TECAN Safire, Research Triangle ark, NC, USA) in fluorescence mode. Excitation was 337 nm with 5 nm scan increments from 400 to 700 nm to produce the spectrum. An integration time of 40s was used for each increment. Imaging of the ejected CHCA particles wa s conducted on an inverted microscope, 100X magnification (Olympus, Melville, NY, USA), using an intensified CCD (Sensicam, IPentamax III, Roper Scientific, Trenton, NJ, USA). A 460 nm bandpass filter was used to select matrix emissi on. Integration time was set at 50 ms. Matrix and Analytes Liquid matrices were prepared by mixing -cyano-4-hydroxycinnamic acid (CHCA) with a liquid support. The liqui d support was equal parts of 50% aqueous ethanol and diethanolamine (DEA). Chromophore concentr ation, CHCA, was 600 mM. To ensure homogeneity and dissolution, th e matrix solution was sonicated for 10-15 minutes. Immediately before analysis the ma trix was vortexed for 10 seconds. For each

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92 analysis, 0.5 L analyte solution and 0.5 L matrix solution were deposited on target, and in the order presented. For direct analysis, analytes were prepared in aqueous 0.1% TFA. Stock solutions of analyte were prepared at 50 pmolL-1, and diluted as noted. All analytes (angiotesin II, bradykinin fragement 1-7, bradykinin, rese rpine, and spiperone), matrix, and solvents were used unpurified (Sigma Al drich, St. Louis, MO, USA). Results and Discussion Liquid Matrix Homogeneity Ion yield inconsistencies in solid matrix MALDI analysis are due to irreproducible sample deposition and laser shot-to-shot variations.152 A liquid sample is homogeneous and provides a reproducible deposition onto the target. While shot-to-shot variations exist with all laser systems, the nitrogen UV laser has only a ~3.5% standard deviation in pulse energy (VSL-337-ND-S, Spectra-Phys ics, Mountain View, CA, USA). The inherent laser variations do not account for the large deviations in analyte ion signals seen with solid matrix MALDI. Instead, the signal fluctuations can be explained by considering the irregularities in crystal forma tion. Some crystals exhibit the optimal ratio of analyte and matrix, thus forming a “sweet spot” for the laser to desorb and ionize analytes. Other crystals may not incor porate analyte—preventing any ion signals. Adding to the irreproducibility of the anal yte ion signals, MALDI spectra, in terms of maximum resolution and mi nimum ion fragmentation, are best obtained slightly above the ion threshold fluence.152 Unfortunately, the maximu m ion yields ar e not obtained until higher fluence is used. Thus, single shot spectra taken at low fluence show low signal intensities and poor reproducibility.

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93 To evaluate the liquid matrix shot-to-s hot reproducibility, sequential laser pulses were analyzed for ion signal variations. Fi gure 4-1 shows five indi vidual laser shots and the corresponding ion signals (using a liquid matrix ). Important in the figure is that each shot produces analyte ions and that successive laser shots are reproducible. 0 5 10 15 20 25 30 0.000.250.500.751.00Time (s)Intensity Bradykinin Fragment TIC Figure 4-1. A plot showing indi vidual ion packets resulting from liquid matrix analysis. The laser was operated at 5 Hz with the mass spectrometer data system collecting spectra at a ra te of 100 Hz. The asynchronous timings of the laser and the pulser allow for temporal profile s of each ion packet to be obtained. To allow for the detection of individual laser pulses, the TOF data system was set at a rate of 100 spectra per second. With the laser pulsing at 5 Hz, time profiles for the ion packets were sampled. Ion-molecule collisi ons at increased pressure spread the initial ion plume to a larger time width, re sulting in an ion packet width ~100 ms.102 Figure 4-1

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94 also shows a slight temporal discrimination of the background (total ion counts) from the analyte ions (bradykinin). The background ions were present after th e analyte ion signal diminished. The ion profile data suggests th at signal-to-noise ratios could be increased by temporally gating the detection syst em, allowing for less background collection between laser pulses. Altera tion of the data system, for this purpose, could not be accomplished without software modificati ons, which were unavailable from the instrument manufacturer. By using the liquid matrix, each laser pulse is able to intera ct with a “fresh” surface. A video of the liquid on the target, Object 4-1, shows surface regeneration with each pulse. Object 4-1. A video file of the laser irradiating the liquid sample is provided. (1.3 mb, Liquidregeneration.mpg, 50 seconds). Laser Frequency Laser frequency may alter the interactions the laser has with the liquid surface. While higher repetition rates introduce more ion packets, it may hinder the surface regeneration process. Inst ead, as the laser frequency was increased, signal variations were reduced as analyte ion si gnals were enhanced. As lase r pulses occur closer together, ion packets began to overlap. The high pressure collisions spread ea ch of the packets to approximately the same width, so the ion sign als were combined in a dense ion beam. A quasi-continuous beam was crea ted allowing for increased an alyte ion signals using a fixed sampling rate. Figure 4-2 shows anal yte ion intensity as a function of laser frequency.

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95 0 100 200 300 400 500 600 0510152025303540 Laser Frequency (Hz)Analyte Intensity150 155 160 165 170 175 180 185Pulse Energy (J) Figure 4-2. A plot of anal yte ion signals and pulse en ergy as a function of laser frequency. The pulser and the la ser were operated asynchronously. Deviations from linearity for the ion signals occurred as pulse energies decreased at higher laser frequencie s. Pulse energies were direct measurements taken before the attenuating optics. Increased frequency allows more ion pack ets to be overlapped, yielding a more intense ion beam. In Figure 4-2, the analyte i on signal begins to incr ease at ~8 Hz. This frequency provides one pulse every 125 ms, co inciding with the beginning of ion packet overlap. A comparison of signal intensities at laser frequencies of 10 Hz and 20 Hz shows a nearly doubled analyte ion signal. At 20 Hz, or every 50 ms, two laser pulses are incorporated into the ion pack et temporal profile of ~100 ms. The 30 Hz frequency does not show this same increase, perhaps explaine d by the reduction in pulse energy at higher frequencies. Figure 4-2 al so contains a secondary y-axis for pulse energy measured as a function of laser frequency. While the la ser specifications rated the system for continuous use at 0-20 Hz, higher repetition rates (up to ~35 Hz) were possible for

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96 limited time periods. There was no mention in the laser manufacturer ’s specifications of variations in pulse energy with incr eased laser frequency. The pulse energy measurements shown in Figure 4-2 explain the non-linear increase in analyte signal intensities with laser frequency—pulse energies are not maintained. Advances in solid-state UV lasers enable the production of co mpact laser systems capable of yielding high repeti tion rates (>1 kHz) with cons istent pulse energies. A recent study has shown that incorporating a hi gh repetition rate lase r into a MALDI TOF instrument provides benefits for high-throughput analysis.160 The oa-TOF used in the current study along with the liquid matrix woul d also benefit greatly from increased laser frequencies. Besides increasi ng analyte ion intensities, the liquid matrix allows a large number of laser shots (>30,000) before the sa mple needs to be replenished or the laser translated. A solid matrix, providing ~ 100 shots per spot and using a 1 kHz laser frequency, would require tran slation every 10 ms. This may be become difficult when considering that each new area in the solid matrix MALDI may or may not provide analyte signals due to po or crystal formation. Fluence Dependence The fluence ranges reported in the literatu re are inconsistent due to measurement inconsistencies for laser spot size.153 Since spot sizes show strong dependence on fluence values, micrometer deviations can yield dr astically different results. An order of magnitude difference in ion fluence thresholds can be seen for measurements made from larger spot sizes ~400 m to smaller spot si zes ~50 m (identical calculated fluence).45 Figures 4-3 and 4-4 show the analyte i on yield dependence of laser fluence for liquid matrices at atmospheric pressure. Fl uence ranges are not c onsidered to be mass dependent; instead, the ranges may be chemical class dependent.152 Figure 4-3 illustrates

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97 the analyte yields for reserpine and spiperone and Figure 4-4 shows th e analyte yields for bradykinin fragment 1-7 and angiotensin II. 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 020406080100120140160180200Pulse Energy (J)Intensity Bradykinin 1-7 AngiotensinII Figure 4-3. A plot of analyte ion yields as a function of pulse energy. The analytes, reserpine and spiperone, were examined at atmospheric pressure using a CHCA chromophore liquid matrix. The laser spot size was approximately elliptical with diameters of 250 m by 300 m. The pulse energies were measured after the attenuating optics. The reporting of pulse energies versus calculated fluence ma y provide a broader insight into the data due to the drastic variati ons in the literature. The calculated fluence ranges for APMALDI with a liquid matrix are 200-2000 Jm-2. In both Figures 4-3 and 44, the spot size was elliptical with diameter s of 250 m and 300 m. Typical ranges for vacuum solid matrix MALDI are 100-1000 Jm-2, with variations reported in the ranges of 30-10,000 Jm-2.45,152

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98 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 020406080100120140160180200Pulse Energy (J)Intensity Reserpine Spiperone Figure 4-4. A plot of analyte ion yields as a function of pulse energy. The analytes, bradykinin fragment 1-7 and angiotensi n II, were examined at atmospheric pressure using a CHCA chromophore liquid matrix. The laser spot size was approximately elliptical with diamet ers of 250 m by 300 m. The pulse energies were measured after the attenuating optics. APMALDI tolerates a larger fluence range while still producing minimal fragmentation of molecular ions.157 This trend holds true for the liquid matrix; however, unlike traditional fluence measurements with vacuum MALDI, lower pulse energy values could not be incorporated. Calibration of the UV neutral density filter wheel (with a variable gradient) showed that slight variations in pulse en ergy were not possible with the optical setup used. Figure 4-5 shows the UV wh eel calibration with direct pulse energy measurements.

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99 0 20 40 60 80 100 120 140 160 180 200 020406080100120140160180Wheel Rotation (degrees)Pulse Energy (J) Figure 4-5. The calibration of a UV neutral density filter wheel for pulse energy. At higher pulse energy ranges, ion yields le veled off or were reduced. This can be attributed partly to increase d fragmentation of the protonate d molecular ions. Figure 4-6 shows reserpine mass spectra obtained at differe nt pulse energies (10, 90, and 180 J). As the pulse energy, or fluence, is increased, fragment ions are produced. Figure 4-7 shows bradykinin fragment 1-7 mass spectra obtained at different pulse energies (10, 90, and 180 J). The peptide shows less structural fragmentation than the small molecule, reserpine, (Figure 4-6) yet both the diethanolamine adduct and protonated molecular ion show increased neut ral water loss. The loss of neutral water from peptides is considered a low energy fragmentation pathway; therefore, even with the highest fluences used only limited fragmen tation occurred with the liquid matrix.161

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100 0 1000 2000 3000 4000 5000 300350400450500550600650700m/zIntensity[M+H]+[M+H]+[M+H]+[M–C10H11O5+H]+[M–C10H11O5+H]+[M-CH3+H]+[M-CH3O+H]+[M-CH3O+H]+[M–C11H14O5+H]+ 10 J/pulse 90 J/pulse 180 J/pulse 0 1000 2000 3000 4000 5000 300350400450500550600650700m/zIntensity[M+H]+[M+H]+[M+H]+[M–C10H11O5+H]+[M–C10H11O5+H]+[M-CH3+H]+[M-CH3O+H]+[M-CH3O+H]+[M–C11H14O5+H]+ 0 1000 2000 3000 4000 5000 300350400450500550600650700m/zIntensity[M+H]+[M+H]+[M+H]+[M–C10H11O5+H]+[M–C10H11O5+H]+[M-CH3+H]+[M-CH3O+H]+[M-CH3O+H]+[M–C11H14O5+H]+ 10 J/pulse 90 J/pulse 180 J/pulse Figure 4-6. Mass spectra illust rating fragmentation produced at higher pulse energies (i.e., 10, 90, and 180 J). The anal yte was 25 pmol of reserpine. Particle Ejection Decreased analyte ion yields shown in Figures 4-3 and 4-4 can not be fully accounted for by increased fragmentation alone. Suggestions have been made that higher fluences produce particle ejections during the MALDI desorption process. Using laser desorption, total particle yi elds have been collected onto a quartz microbalance as a function of laser fluence.162

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101 0 1000 2000 3000 4000 5000 6000 200300400500600700800900100011001200 m/zIntensity[M+H]+[M+K]+[M+H]+[M+H]+[M+Na]+ [M+2DEA+H]+[M+2DEA+H2O+H]+ 10 J/pulse 90 J/pulse 180 J/pulse 0 1000 2000 3000 4000 5000 6000 200300400500600700800900100011001200 m/zIntensity[M+H]+[M+K]+[M+H]+[M+H]+[M+Na]+ [M+2DEA+H]+[M+2DEA+H2O+H]+ 0 1000 2000 3000 4000 5000 6000 200300400500600700800900100011001200 m/zIntensity[M+H]+[M+K]+[M+H]+[M+H]+[M+Na]+ [M+2DEA+H]+[M+2DEA+H2O+H]+ 10 J/pulse 90 J/pulse 180 J/pulse[M-H2O+H]+ [M-H2O+H]+ [M-H2O+2DEA+H]+ Figure 4-7. Mass spectra illust rating fragmentation produced at higher pulse energies (i.e., 10, 90, and 180 J). The analyte wa s 25 pmol of bradykinin fragment 17 The study, done in vacuum, reports neutra l molecules begin desorbing at 11 mJcm-2. Alves, Kalberer and Zenobi examined charge d particle ejections from MALDI matrices at atmospheric pressure.154 They reported that the part icles ejected were, on average, larger that those for vacuum MALDI. Es timates for the particles ranged from ~110 to 240 nm, depending on the matrix preparation and laser fluence. The APMALDI study also determined that as fluence increased, mean particle size also incr eased. Still it was deemed difficult to determine the contributi on of particles produced by the laser ablation process versus the gas-phase processes occurr ing in the MALDI plume. Examination of liquid matrices under vacuum has also shown th at increases in fluence can produce larger particle sizes.162

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102 To provide an explanation for decreasing ion yields at higher fluence and obtain values for liquid matrices at AP, particle ejections were examined. By collecting fluorescence images of the liquid matrix duri ng laser desorption, particles that were ejected could be imaged. Since the MALD I matrix requires a UV chromophore for the desorption and ionization processes to occur, an imaging method is inhe rent in the liquid matrix. Figure 4-8 shows the emission spectrum for the CHCA liquid matrix. Excitation occurred using 337 nm, the wavelength used fo r desorption/ionization. Table 4-1 lists the conditions used for the emission spectrum collection. 0 10000 20000 30000 40000 50000 400450500550600650700Wavelength (nm)Intensity (counts) Figure 4-8. Emission spectrum collected fo r the CHCA liquid ma trix using a 337 nm excitation wavelength.

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103 Table 4-1. Conditions used to acquire the emission spectrum of the CHCA liquid matrix. Measurement Mode Fluorescence Top Excitation wavelength 337 nm Emission wavelength start 400 nm Emission wavelength end 700 nm Emission scan number 61 Emission wavelength step size 5 nm Excitation bandwidth 12 nm Number of flashes 10 Integration time 40 s A 460 nm bandpass filter, a gated intensified CCD, and an inverted microscope (100X objective) were used to collect fluor escence images of the liquid matrix. The excitation source was the same 337 nm nitrogen laser used in the APMALDI configuration. Figure 4-9 shows th e laser spot on the liquid matrix. Collecting 50 ms images at varied fluences allowed videos of the particle ejection process to be constructed. Figure 4-10 show s a still image of the particles ejected at higher fluences (pulse energy 180 J) using the liquid matrix.

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104 100 m Liquid droplet Laser spot Figure 4-9. Fluorescence image of the laser impinging on the liquid matrix surface. The spot size can be estimated at ~300 m in diameter. Only approximate values for the number of pa rticles ejected and th e particles sizes are available. The images are obtained in a depth of focus field; therefore, only a slice of the particles ejected could be viewed. Also, th e isotropic emission of the CHCA particles provides only a rough estimate of particle size, hundreds of na nometers. Important in the study is the dependence of particle ej ection on pulse energy (and correspondingly fluence). Below 90 J pulse energy, partic le ejection was errati c. Increasing pulse energies yielded more frequent ejection of pa rticles. Fluorescence images collected at higher fluences showed particles ejected from the liquid matrix. Videos showed that the particle ejections occurr ed at pulse energies of 110, 140, and 180 J.

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105 100 m Figure 4-10. Fluorescence image of particles ejected from the liquid matrix. The pulse energy was ~180 J. Object 4-2. A video of the laser irradiat ing the liquid sample demonstrating particle ejection at 110 J pulse energy. (1 mb, 110pulseenergy.mpg, 10 seconds). Object 4-3. A video of the laser irradiat ing the liquid sample demonstrating particle ejection at 140 J pulse energy. (1 mb, 140pulseenergy.mpg, 10 seconds). Object 4-4. A video of the laser irradiat ing the liquid sample demonstrating particle ejection at 180 J pulse energy. (1 mb, 180pulseenergy.mpg, 10 seconds). Object 4-5. A video of the laser irradiat ing the liquid sample demonstrating particle ejection at 180 J pulse en ergy—magnified view. (1 mb, 110pulseenergyzoomed.mpg, 10 seconds). Object 4-6. A video of the laser irradiat ing the liquid sample demonstrating particle ejection at 180 J pulse energy—slow motion (non-false color) view. (5.5 mb, 180pulseenergyslowed.mpg, 50 seconds).

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106 Plume Interactions Fluence ranges play a major role in th e desorption processes for MALDI, yet interactions in the ejected pl ume also have important mechanistic consequences. Signal suppression illustrates possible process pathwa ys. Two suppression effects are known to occur: the matrix suppression effect and the analyte suppression effect.163-164 The matrix suppression effect occurs when a sufficiently high analyte concentration suppresses matrix ion signals in MALDI spectra When excited matrix molecules are in close proximity to the analyte, reactions can transfer energy and charge. Analyte signal suppression effects are also rela ted to plume interactions. Sp ecific analyte ion signals are reduced or increased due to their chemical properties and the ion-mo lecule interactions that occur in the MALDI plume. Since the liquid matrix provides a homogeneous environment, varying analyte molar ratios wi th peptide mixtures can demonstrate the analyte suppression effect (i.e., plume interactions). Preliminary experiments demonstrating this phenomenon in the liq uid matrix were conducted by analyzing equimolar peptide mixtures. Figure 4-11 s hows the ion signals obtained for bradykinin fragment 1-7 and angiotensin II. A comparison of Figure 4-11C with Figures 4-11A and 4-11B shows that ion signal intensities are not maintained. While the individual ion signals are not equivalent in most mixtures, due to ionization differences, th e total peptide signal is also reduced dramatically. At a specific fluence (90 J) a limited number of excited matrix molecules are emitted during desorption. These molecules undergo ion-molecule reactions in the plume. A limited number of excited molecu les can only produce a specific amount of analyte ions—in this case only a fracti on of each analyte in the mixture.

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107 0 1000 2000 3000 4000 5000 6000 7000 8000 70075080085090095010001050110011501200m/zIntensity 0 1000 2000 3000 4000 5000 6000 7000 8000 70075080085090095010001050110011501200m/zIntensity 0 1000 2000 3000 4000 5000 6000 7000 8000 70075080085090095010001050110011501200m/zIntensityangiotensinII bradykinin 1-7 Equal molar peptide mixture [M1+H]+[M2+H]+[M1+H]+[M2+H]+[M1+Na]+[M1+K]+[M2+Na]+[M2+K]+[M2+DEA+H]+[M2+Na]+A B C Figure 4-11. Mass spectra demonstrating analyt e signal suppression are shown. The three spectra represent A) angiotensin II, B) bradykining fragment 1-7, and C) the combined analysis of both peptides. For the single peptide analyses 25 picomoles of each was placed on target. For the combined analysis 25 picomoles of each peptide was placed on target. Conclusions Atmospheric pressure liquid matrices can add analytical utility to mass spectrometry. With the init ial studies for formulating liquid matrices completed, the focus shifted to characterizing desorption and ionization in liquid systems. Though viscous liquids have been examined in the vacuum chamber, relatively few studies have evaluated volatile matrices at AP.

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108 This chapter evaluated UV liquid matrices at atmospheric pressure with respect to laser parameters. Using liquid matrices, ion yields and particle ejection were examined versus laser fluence adjustments. Fluence dependence is both matrix preparation and pressure dependent, so the optimal laser flue nce needed to be determined for the liquid matrix preparation and sampling pressure used. Additionally, sampling rate becomes important when evaluating high-throughput tech niques. Since liquid matrices provide regenerating surfaces and longer sampling tim es, the common nitrogen laser repetition rates, <20 Hz, have become a limitation. While the details of ion formation in MALDI are still undetermined, parametric studies relating laser variable s to analyte signals must be conducted for application development. The atmospheric pressure liqui d matrix can be sampled at higher fluences before fragmentation and partic le ejection decrease analyte i on yields. While ion yields can be related directly to laser parameters MALDI plume interacti ons also affect ion intensities. Although the liquid matrix is homogeneous, the sample composition and sampling parameters dictates matrix and analyte ion populations.

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109 CHAPTER 5 FUNCTIONALIZED NANOPARTICLES FOR LIQUID APMALDI PEPTIDE ANALYSIS Introduction Koichi Tanaka during his 2002 Nobel lecture summarized the thoughts of numerous MALDI researchers, demonstrating what is known, not known, and what most of the scientific community does with the information.165 “Herein I have suggested the principles by which laser-light irra diation is able to generate huge molecular ions. However, these principles are not necessarily correct because they have not yet been fully proven scientifically….even if the principles are unverified, their application takes priority if they are useful and practical.” —Koichi Tanaka 2002 With its ability to ionize and meas ure large biopolymers, biological mass spectrometry is a routine analysis tool. However, as samples become more complex and the analytes less abundant, methods for selec tive extraction and preconcentration must be developed. The recent instrumental developm ent of atmospheric pressure matrix-assisted laser desorption/ionization (APMALDI) offers an advantage for biological analysis.103,108,114,143,166 Atmospheric pressure (AP) sampling encourages alternative sampling approaches, such as simple UV abso rptive liquid matrices that allow rapid and reproducible peptide analysis.168 This chapter describes a new AP liquid matrix method that incorporates functionaliz ed silica nanoparticles as scav enging agents for peptides. For complex biological samples, analysis time is limited less by the mass analysis steps than by the precursor separation proce sses. In an effort to decrease separation

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110 procedures prior to mass spectrometry analys is, various techniques are employed. In offline systems, solid phase extraction tools, such as Ziptips (Millipore, Billerica, MA, USA), are often used for the removal of interferences from biopolymer samples.169 This solid phase extraction (SPE) procedure incl udes a multiple step process for analyte adsorption and extraction. While the procedure does allow sample cleanup and preconcentration, it does not provide a means for selective analyte removal. Another approach, loosely adapted from SPE, is su rface enhanced laser/de sorption ionization (SELDI).170 In SELDI, a MALDI target plate is functionalized for the selective retention of analytes. The surface functionalization a llows analyte retenti on while removing mass spectrometry interferences through on-target washing. SELDI offers increased throughput for applications in biological sy stem profiling; however, the technique currently requires the use of a vacuum chamber, n ecessitating a delay between analyses.171 As an alternative, discrete particles can be used for the removal of analytes prior to MALDI analysis. The range of material properties available provides choice in functionalization chemistry and extraction pro cedure. Previously, commercially available micrometer-sized particles have been used in conjunction with vacuum MALDI.172-173 In this approach, nonselective interactions were used to concentrate analytes for conventional MALDI analysis. Recently, the affinity capture of analytes using magnetic micrometer-sized particles has been presented by Tempst et. al. These experiments also were conducted using traditional vacuum MALDI analysis.174-175 While silica based nanoparticles have been used as analyte recognition elements, their application to mass spectrometry anal ysis has not been extensively explored.176-177

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111 We propose using them as a simple and rapid means for sample preparation for liquid APMADLI. Liquid matrices, which serve well for atmospheric pressure analysis, can also act as a medium for the functionalized nanoparticles. Liquid matrices alleviate the need for sample vacuum chambers and a void the well known inhomogeneities associated with crystalline matrices. C18 nanoparticle s (silica nanoparticles f unctionalized with 18 carbon hydrophobic chains) are shown to extract va rious standard peptides from solutions and allow a rapid analysis by directly anal yzing the particles with liquid APMALDI. While the C18 nanoparticles utilize hydrophobi c interactions to facilitate analyte extraction, we have also investigated apta mer based nanoparticles and their ability to selectively isolate specific peptides. Thes e take advantage of th e affinity of singlestranded nucleic acids for peptide molecules. Aptamers are typically between 15 and 70 nucleotides in length and form elaborate three-dimensional structures and shapes, allowing the aptamers to have high selectivity an d affinity for a wide range of molecules. Aptamers have been well documented in ar eas such as investigating cellular protein functions and protein/ ligand interactions.178-180 Selective analyte removal is often done using antibody-antigen interactions in bioana lysis procedures; howev er, while antibodies provide selectivity through c onformational binding sites and a low dissociation constant (Kd), yielding low detection limits, extensive characterization and in vivo production are required.181-182 As an alternative to antibodies aptamers have several inherent advantages. Since they consist of a short, single strand of DNA or RNA, they are less costly to synthesize and have a longer shelf life. The aptamer selection process mimics natural selection, so in theory it is possibl e to develop a highly specific aptamer for any target molecule. In comparison to engineeri ng antibodies for particular applications, the

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112 incorporation of site-specific labels or coupl ing sites into an aptamer is a less complex process. In addition, aptamers are synthesized completely in vitro, allowing for the development of high throughput selection methods.183-186 The aptamer used in this study was previously selected for D-vasopressin (Cys1-Tyr-Phe-Gln-Asn-Cys6-Pro-Arg-GlyNH2) and has a binding efficiency of ~1000 fold over L-vasopressin, as seen in studies conducted using the aptamer as a chiral se paration group for affinity chromatography.187188 Nanoparticles for the extraction of pep tides and subsequent analysis using atmospheric pressure matrix-assisted laser desorption/ionization (A PMALDI) have been evaluated. The atmospheric pressure source allo ws particles to be directly introduced in the liquid matrix, minimizing sample loss and an alysis time. Described in this chapter are two sample preparation procedures for liquid APMALDI analysis: a C18 functionalized silica nanoparticle fo r hydrophobic extractions, and an aptamer functionalized magnetite core nanoparticle fo r rapid, affinity extractions. The C18 particles provide a non-selective support fo r rapid profiling applications, while the aptamer particles are directed towards reducing the complexity in biological samples. The aptamer functionalized particles provide a more selective analyte-nanoparticle interaction whereby the tertiary structure of the analyte becomes more critical to the extraction. In both cases, the liquid APMALD I matrix provides a support for ionization, and acts as the releasing agent for the analyteparticle interaction. Additionally, analyte enrichment was possible due to the large surf ace to volume ratio of the particles. The experiments conducted with functionalized nano particles, in an atmospheric pressure

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113 liquid matrix, present a basis for furt her methodologies and utilities of silica nanoparticles to be developed. Experimental Methods Materials Peptide standards (Sigma-Aldrich Corp., St. Louis, MO, USA), angiotensin I, angiotensin II, bradykinin, bra dykinin fragment 1-7, and L-va sopressin were prepared as stock solutions of 500 pmolesL-1 using either acetonitrile (A CN), for C18 nanoparticle extractions, or water, for aptamer na noparticle extractions. D-vasopressin (Genomechanix, Gainesville, FL, USA) was synthesized using conventional fluorenylmethoxycarbonyl chemistry, and dissolv ed in water for analysis. Biotynalated DNA was also purchased from Genomechanix (Gainesville, FL USA). Non-extraction analysis was conducted by spotting 0.5 L of matrix onto 0.5 L of analyte stock solutions. Nanoparticle Synthesis Silica C18 functionalized nanoparticles The silica C18 functionalized nanoparticle s were prepared using a previously reported synthesis procedure.189-190 Briefly, the nanoparticle s were prepared using a water-in-oil (W/O) microemulsion with a water-to-surfactant molar ratio of 10:1. The synthesis produced uniform silica nanoparticles (60 +/5 nm in diameter). Twenty hours into the synthesis, 40 L of octadecyltrimethoxysilane (Sigma -Aldrich Corp., St. Louis, MO, USA) and 10 L of ~30% ammonium hydr oxide (Fisher Scientific, Fair Lawn, NJ, USA) were added to the microemulsion. The mixture was stirred for an additional 4 hours to yield a C18 outer coating of the si lica core nanoparticles. Prior to peptide extraction, the nanoparticles were washed w ith ethanol, acetone, and water three times

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114 each, and redispersed in acetonitrile. The final concentration of the nanoparticle suspension was approximated at ~6 mgmL-1. Magnetic aptamer nanoparticles The iron oxide core magnetic nanopartic les were prepared using the Stber method.191 The magnetite core was formed by precipitating iron oxide through mixing ammonia hydroxide (2.5%) and iron chloride at 350 RPM using a mech anical stirrer (10 minutes). The iron chloride solution cont ained ferric chloride hexahydrate (0.5 M), ferrous chloride tetrahydrate (0.25 M), and HCl (0.33 M).192 After three washes with water and once with ethanol, an ethanol so lution containing ~1.2% ammonium hydroxide was added to the iron oxide nanoparticles, yiel ding a final concentration of ~7.5 mgmL-1. Tetraethoxyorthosilicate (200 L) was adde d to create the silica coating for the magnetite core particles. The mixture was sonicated for 90 minutes to complete the hydrolysis process, and the nanoparticles we re washed three times with ethanol to remove excess reactants. Aptamers were immobilized onto the partic le surface through avidin-biotin linkage (5' – biotin-TCACGTGCAT GATA GACGGC GAAGCCGTCG AGTTGCTGTG TGCCGATGCA CGTA).193 For avidin coating, a 0.1 mgmL-1 Fe3O4-si (silica coated magnetic nanoparticles) solution and a 5 mgmL-1 avidin solution were sonicated in the presence of the particles for 5 minutes and in cubated at 4 C for 14 hours. The particles were magnetically separated and washed three times with 10 mM phosphate buffered saline (PBS) pH 7.4. The particles were redispersed at 1.2 mgmL-1 in 10 mM PBS and stabilized by cross-linking the coated nanopart icles with 1% glutaraldehyde (1 hour at 25 C). After another separation, the particles were washed three times with 1M Tris-HCl buffer and spun for 10-15 minutes at 14,000 RPM. For aptamer attachment, the particles

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115 were dispersed at 0.3 mgmL-1 in 20 mM Tris-HCl, 5 mM MgCl2, pH 8.0. Biotin labeled DNA was added to the solution at a concentration of 0.2 x10-6 M. The reaction was incubated at 4 C for 12 hours. Three final washings of the partic les were done using 20 mM Tris-HCl, 5 mM MgCl2 at pH 8.0. The nanoparticles were made to a final concentration of ~0.3 mgmL-1 and stored at 4 C before use in the same buffer. Matrix and Analyte Preparation The liquid matrix was a UV absorbing formulation developed for use with APMALDI. The matrix was prepared by mixing -cyano-4-hydroxycinnamic acid (CHCA) (Sigma-Aldrich Corp., St. Louis, MO, USA) with a liquid support containing a solvent liquid, equal parts ethanol and water (Fisher Scientific, Fair Lawn, NJ, USA), and a viscous component, dietha nolamine (DEA) (Sigma-Aldri ch Corp., St. Louis, MO, USA). The matrix was sonicated an d vortexed to ensure dissolution. Instrumentation The mass spectrometer used was an or thogonal-acceleration time-of-flight mass spectrometer (oa-TOFMS) (LECO Corporation, St. Joseph, MI, USA). Details of the mass spectrometer and source are described in Chapter 2. During analysis, the laser was pulsed (20 Hz) asynchronously with the MS repeller pulse (5 kHz). Spectra were exported and stored in an exte rnal computer at a rate of ~4 spectra per second. The spectra shown are an accumulation of summed spectra for 1-5 minutes. Fluorescence measurements for the extrac ted fluorescein labeled angiotensin II (Sigma-Aldrich Corp., St. Louis, MO, USA) were made using a microplate reader (TECAN Safire, Research Triangle Park, NC, USA) in an epi-illumination mode with excitation at 485 nm (5 nm bandwidth) and collection at 520 nm (5 nm bandwidth). Imaging of the C18 nanoparticles was c onducted on an inverted microscope, 100X

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116 magnification, (Olympus, Melville, NY, USA) using an intensified CCD (IPentamax III, Roper Scientific, Trenton, NJ, USA). Tr ansmission electron micrographs (TEM) of nanoparticles were taken using a 7200 H itachi transmission electron microscope. Extraction Procedures Two procedures were used for peptide ex tractions: a centrifug ation technique was applied for the silica C18 particles, while the magnetic particles required only a magnetic separation. The process of ex traction for the C18 particles is demonstrated in Figure 5-1. 90 L 10 L supernatant 100 L Wash solution Centrifuge + + Centrifuge Vortex & +supernatant +Liquid Matrix Mass spectrum Analyte Nanoparticles Mass spectrum Figure 5-1. An illustration of the centrifugation technique used for nanoparticle extractions is shown. For the C18 functionalized particles, 90 L of nanoparticle solution (~4 x 10-8 M) was incubated with 10 L of stock analyte so lution (~1:1000 particle -to-analyte ratio) for 10 minutes. The mixture was centrifuge d for 5 minutes at 14,000 RPM (Eppendorf 5180R, Fisher Scientific, Fair Lawn, NJ, USA) After centrifugation, the supernatant was

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117 removed leaving ~1 L of silica particles. The particles were washed to remove interferences and unabsorbed analyte. The wash solutions, 99 L of acetonitrile or water, were added and the mixture was vortexed ~5 seconds. The wash solution used was dependent upon the desired resu lt, analyte removal or rete ntion. The procedure was repeated when additional washing steps we re necessary. For nanoparticle extraction analysis, the silica particles remaining in the centrifuge tube after supernatant removal were pipetted directly to the MALDI ta rget surface as shown in Figure 5-2. Liquid Matrix Analysis Extracted particles Nanoparticles Figure 5-2. A diagram illustrating the utility of the nanoparticles with the liquid matrix is shown. The aptamer functionalized magnetic nanopa rticles allowed rapid separation using a simple magnetic extraction method rather than centrifugation. The simplified extraction procedure for the magnetic na noparticles is shown in Figure 5-3A. Additionally, a depiction of the functionalized particles is shown as Figure 5-3B.

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118 90 L 10 L supernatant + + +Liquid Matrix Mass spectrum Analyte Nanoparticlesmagnet [Arg8]Vasopressin H-Cys1-Tyr-Phe-Gln-Asn-Cys6-Pro-Arg-Gly-NH2 [Arg8]Vasopressin H-Cys1-Tyr-Phe-Gln-Asn-Cys6-Pro-Arg-Gly-NH2B A Mass spectrum Figure 5-3. An illustration of the magnetic separation technique used for nanoparticle extractions is shown. Inset in the figure is representation of the aptamer functionalized magnetic nanoparticles. First, a buffer exchange was conducted using 50 L of aptamer conjugated nanoparticles (~1 x 10-8 M). The particles are remove d from solution using a neodymium iron boron magnet (12,200 gauss, Edmund Optic s, Barrington, NJ, USA) and washed with 5 mM phosphate buffer and then 3 mM MgCl2, pH 6.0. The buffer used was based upon the reported protocol for aptamer ch iral separation of L and D vasopressin.187-188 The resulting nanoparticle suspension was used in analyte extractions For extraction, 10 L of 50 pmolL-1 analyte solution was incubated with 50 L of the nanoparticle suspension for 10 minutes (~1:100 particle-toanalyte ratio). A magnet directed the particles to the bottom of the vial for removal of the supernatant. The remaining particles, ~1 L, were mixed with 5 L of wa ter and 5 L of liquid matrix. Analysis was

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119 conducted by pipetting 0.5 L of the apta mer particle/matrix mixture on the MALDI target. Results and Discussion Background Atmospheric pressure matrix-assisted laser desorption/ionization offers the advantage of analyzing samples at ambient conditions. Limiting the vacuum constraint of a MALDI source allows analysis proce dures to be developed using non-vacuumcompatible matrices. As the complexity of biological samples increases, simple methods that limit the necessary separation procedur es will be valuable for biological mass spectrometry analysis. Nanoparticle Characterization Two particle types were examined for use with liquid APMALDI: a C18 functionalized nanoparticle for hydrophobic extr action, and an aptamer functionalized magnetic nanoparticle for affinity extracti ons. These silica base d nanoparticles were developed to aid and simplify sample prepar ations for liquid APMALDI applications. The C18 coated nanoparticles provide a support for rapid profiling applications while the aptamer nanoparticles are dire cted towards reducing the complexity in biological samples. In separation applications using si lica coated nanoparticle s, particle dispersion is a major concern.179 One method to combat particle aggregation, and allow efficient analyte binding, is surface modifications.192 The use of C18 provides hydrophobic binding for extractions while disper sing the particles in acetonitrile.

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120 To explore extraction possibili ties for the C18 silica partic les, both size distribution and peptide attachment were examined prio r to conducting mass spectrometric analysis. Nanoparticle characterization is shown in Figure 5-4. A 50 nm 50 nmB DExcitation: 485 +/-5 nm Emission: 520 +/-5 nm 0 10000 20000 30000 40000 50000Reaction Mixture Wash1 Supernatant Wash2 Supernatant Extracted NanoparticlesFluorescence Intensity 100 nmC Figure 5-4. Nanoparticle charac terization using fluorescence an alysis is shown. Figure 54A is a TEM of the C18 functionalized silica nanoparticles. Figure 5-4B is a TEM of the D-vasopressin aptamer f unctionalized magnetic nanoparticles. Figure 5-4C is a fluorescence image of the silica nanoparticles with FITC labeled angiotensin II. Figure 54D shows the fluorescence intensity measured for the extraction and washing steps for FITC labeled angiotensin II with the C18 functiona lized nanoparticles. Figure 5-4A is a transmission electron mi crograph (TEM)of the C18 functionalized silica nanoparticles. Figure 5-4B shows a TEM of the magnetic nanoparticles (~30 nm) with silica coating and aptamer attachment. The particles are well dispersed and have

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121 an average size of ~60 nm. The D-vas opressin aptamer functionalized magnetic nanoparticles are ~30 nm in diameter as can be seen from Figure 5-4B. They are less dispersed due to aggregation. Before liqui d APMALDI was performed, the ability of the C18 nanoparticles to extract peptides from a solution was investigated with fluorescence microscopy and spectroscopy. Figure 5-4C shows a fluorescence image for an isothiocyanate fluorescein (FITC) labe led angiotensin II peptide bound to a C18 functionalized silica nanoparticle. The labe led peptide was mixed, via the extraction procedure described previously (see Experiment al), with the nanopartic les to determine if peptide adsorption occurred on the particle su rface. The image demonstrates peptides can bind to the particle surface of the pa rticle. Figure 5-4D shows the fluorescence intensity measured by a plate reader for each so lution of the peptide ex traction procedure. The initial reaction solution has minimal fluor escence after the extraction has taken place. Subsequently, the two washing steps also have minimal fluorescence after removing the particles. The minimal fluorescence signal fo r the reaction mixture, after the removal of the particles, shows an efficient extraction ha s occurred. The reten tion of the peptide on the nanoparticle after two washing steps is appa rent from the prominent signal in the final nanoparticle extraction and no signal present in the washing solutions. Only ~3% of the total fluorescence signal remained in the mixt ure supernatant. Th e C18 functionalized nanoparticles offer nonspecific binding through van der Waals interactions; therefore, analytes bind through an adsorption method. The fluorescence results suggest that the C18 silica nanoparticles can extract peptide molecules from solutions using simple hydrophobic interactions.

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122 The binding characteristics of the ma gnetic silica nanoparticles have been previously characterized.179,192 For the avidin coated nanoparticles fluorescence DNA hybridization studies show approximately 30 aptamer molecules per magnetic nanoparticle. The aptamer molecules atta ched were estimated through fluorescence measurements based on the amount of complimentary DNA hybridized to the immobilized DNA on the particle surface. Peptide Analysis by Liquid AP MALDI with C18 Nanoparticles The C18 functionalized nanopa rticles allowed the extract ion of a fluorescently labeled peptide. To demonstrate mass sp ectrometric analysis, three peptides – angiotensin I, angiotensin II, and bradykinin fragment 1-7 – were extracted from solution and analyzed using liquid APMALDI. Crystall ization of the matrix was not necessary. Instead, the particles we re introduced directly into the li quid for analysis, yielding a rapid extraction and analysis procedur e. Figure 5-5 shows a chart of normalized ion signals for each peptide’s m/z during each successive step in the extraction process. Five extractions for each peptide were c onducted. Limited analyte signal is seen for all acetonitrile washes, demonstrating the nanoparticles’ ability to extract biopolymers and then release them for MS analysis. The mixture supernatant ion signal can be attributed to overloading of peptide for each particle (1:1000 partic le-to-analyte ratio); therefore, it is assumed the maximum numb er of analyte molecules was bound to the particle surface. In comparison to the fluor escence extraction data of the FITC labeled angiotensin II, the label may a lter the nanoparticle binding char acteristics. The increased hydrophobicity of the FITC label versus the non-labeled peptide is suggested by the decreased peptide extraction for mass spect rometry analysis in Figure 5-5. The extraction procedure was not optimal; howev er, the relationship between the mixture

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123 supernatant signal and the na noparticle suspension signal hints to the nanoparticle capacity. 0102030405060708090100 Reaction Supernatant Wash 1 Supernatant Wash 2 Supernatant Extracted Nanoparticles Normalized Intensity bradykinin angiotensinI angiotensinII 0102030405060708090100 Reaction Supernatant Wash 1 Supernatant Wash 2 Supernatant Extracted Nanoparticles Normalized Intensity bradykinin angiotensinI angiotensinII Figure 5-5. A chart of the pr otonated molecular ion mass sp ectrometry signals for three peptides is shown. Five extractions for each peptide were conducted using C18 functionalized silica nanoparticles. Extraction analysis included mass spectra for the reaction mixture afte r the particles were removed, the supernatants after each wash step and the nanoparticles after the final extraction. Washes were conducted using acetonitrile. The peptide binding capacity for the na noparticles is currently being evaluated using quantitative techniques. Analysis of the extracted and supernatant ion signals in Figure 5-5 may give some insight to the capac ity, but additional qua ntitative studies will provide more definitive basis. This fi gure also demonstrates the ability of C18

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124 functionalized nanoparticles to be used dire ctly in a liquid APMALDI matrix, where the matrix acts as the releasing agent an d allows for ionization to occur. Figures 5-6A, 5-6B, and 5-6C, show sp ectra collected from the nanoparticle suspensions for angiotensin I, angiotensin II, and bradykinin of Figure 5-5 (extracted nanoparticles). 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 1280128512901295130013051310 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 1280128512901295130013051310 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 10601065107010751080 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 10501055106010651070A B C 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 1280128512901295130013051310 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 1280128512901295130013051310 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 10601065107010751080 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 10601065107010751080 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 10501055106010651070 0 200 400 600 800 1000 1200 0500100015002000250030003500 m/zNormalized Intensity 10501055106010651070A B C Figure 5-6. Mass spectra collected from nanopa rticle extractions. Figures 5-6A, 5-6B, and 5-6C show spectra collected for th e nanoparticle extraction of angiotensin I, angiotensin II, and bradykinin, respectively. Protonated molecular ions, as well as s odium adducts, are present indicating the nanoparticles do not interfere with the APMALDI process. Minimal background in the low m/z range is generated with the nanoparticle s in the liquid suspension. This fact becomes important when analyses of lower molecular weight components are analyzed

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125 (e.g., peptide fragments). On the other hand, the increase in adduct formation is most likely attributed to the nanopart icles. The porous silica nanopar ticles allow entrapment of sodium during the synthesis procedure. Wa shing the nanoparticles with ethanol, acetone, and water before redispersing them in acetonitr ile significantly reduced adduct formation, but does not eliminate it. Using peptide sp iked nanoparticle suspensions, reductions in molecular ion suppression and adduct formati on with subsequent water washings were monitored, as shown in Figure 5-7. 0 500 1000 1500 2000 2500 3000Counts Nanos + MatrixPeptide + MatrixNanos + Peptide + Matrix Washed Nanos + Peptide + Matrix Supernantant + Peptide + Matrix 2nd Washed Nanos + Peptide + Matrix M+H 1 Na 2 Na 3 Na Total counts Figure 5-7. A chart showing the effect multip le washing steps has on the sodium adduct signals during mass spectrometry analysis. Both the total and the molecular ion signal doubled from the first water wash to the second. The additional removal of salts can be done with cation-ex change resin beads, volatile ammonium salts such as ammonium citrate or ammonium acetate, and on-probe purification using nitroc ellulose or modified Nafion film substrates.108 While adducts were present for each analysis, the mass spectra of the peptides were not otherwise

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126 adversely affected, as can be seen from the isot ope ratios in Figure 5-6. Direct analysis of the nanoparticles in the liquid matrix lim its analyte loss and provides the opportunity to concentrate the analyte. The nanopartic les have a unique advantage when they are used as a solid phase extraction support. The larger surface area to volume ratio can allow maximum peptide attachment in a mi nimum volume. A preconcentration of the analyte can occur by drawing peptide into a limited analysis volume. Because APMALDI is a mass sensitive technique, pulli ng all the analyte into a small sample volume allows the entire sample to be analyz ed on target. To demonstrate, Figure 5-8 shows the analysis of a 1 M angiotensin II solution before a nd after nanoparticle extraction. Analysis was conducted by placi ng 0.5 L of sample on target and then adding 0.5 L of liquid matrix, ~500 femt omoles on target. Figure 5-8A has the molecular ion peak for angiotensin II before preconcentration and figure 5-8B shows the mass spectrum of the analyzed nanoparticles. 0 50 100 150 200 250 300 700800900100011001200130014001500 m/zIntensity 0 50 100 150 200 250 300 700800900100011001200130014001500 m/zIntensity[M+H]+[M+H]+AB 0 50 100 150 200 250 300 700800900100011001200130014001500 m/zIntensity 0 50 100 150 200 250 300 700800900100011001200130014001500 m/zIntensity[M+H]+[M+H]+AB 0 50 100 150 200 250 300 700800900100011001200130014001500 m/zIntensity[M+H]+[M+H]+AB Figure 5-8. Mass spectra collected for a 1 M angiotensin II solution before and after nanoparticle extraction are shown. Fi gure 5-8A shows the spectrum for the analysis of 0.5 L of the solution, ~500 femtomoles on target. Figure 5-8B shows the spectrum for the analysis of 0.5 L of the nanoparticle extract.

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127 For extraction, a 1 mL solution of 1 M a ngiotensin II was combined with 30 L of C18 functionalized nanopart icles. After one acetonitrile wash, the extracted nanoparticles were redispersed in 5 L of acetonitrile, of which 0.5 L was used for analysis. A comparison of the two spectra s hows ~15 fold concentration enhancement of the analyte. More select ive analyte-nanoparticle interactions may permit wider applications. Concentrating the analyte may improve analysis when low amounts of analyte are present. Previous biopolymer extractions coupled to MALDI remove the analyte from the support before analysis; however, our method inco rporates the particle directly into the liquid matrix solution to alleviate sample transfer losses and reduce preparation time.194 The matrix acts as the releasing agent to allo w for analyte detection. Additionally, the increased surface area to volume ratio of the particles in comparison to micrometer-sized particles can increase extraction efficiency yielding more analyte per sampling volume. Using C18 functionalized supports, adsorpti on and removal of analyte is conducted through mobile phase flow. The molecules w ith limited retention en ter the mobile phase and are eluted more rapidly. Applying this principle to nanoparticle applications, multiple washings were conducted with an ex tracted peptide mixture. Retaining and removing analytes using separa tion techniques allows for detection at each stage of a multiple step washing procedure. To demonstrate, a mixture of three peptides, GLYTYR, VAL-TYR-VAL, and angiotensin II, wa s incubated with nanoparticles to examine retention and release. Figure 5-9 shows the peptides ion signals for each step in the washing procedure.

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128 Diand tripeptides are not re tained on typical C18 solid supports in reverse-phase chromatography due to their limited hydrophobicity ; therefore, these along with a large peptide were used to examine retention order.195-196 0 10 20 30 40 50 60 70 80 90 100Normalized Intensity Reaction Supernatant Wash 1 Supernatant Wash 2 Supernatant Extracted Nanoparticles GLY-TRY VAL-TRY-VAL angiotensinII 0 10 20 30 40 50 60 70 80 90 100Normalized Intensity Reaction Supernatant Wash 1 Supernatant Wash 2 Supernatant Extracted Nanoparticles GLY-TRY VAL-TRY-VAL angiotensinII Figure 5-9. A chart of the pr otonated molecular ion mass sp ectrometry signals for three varied length peptides is shown. The sm allest peptide is retained the least, while the largest peptide remain s on the particle. The hydrophobic interactions are minimized with small pe ptides, so retention of the analyte on the particles is limited. Washes were conducted using acetonitrile. Each peptide differs in its ability to bind to the C18. Using only acetonitrile washes, the smallest peptide was removed first (lowest retention time). The largest peptide, angiotensin II, is more hydrophobic and is retained after both wa shes. Also evident is the order the two smaller peptides are rem oved, GLY-TYR then VAL-TYR-VAL. While this process remains a low resolution sepa ration mechanism, the elution order of the peptides correlates with hydrophobicity. The cap ability of controlled release adds to the

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129 utility of these particles in biological mass spectrometry analysis in situations where hydrophobic properties as well as qualit ative identification are important.197 Peptide Analysis by Liquid APMA LDI with Aptamer Nanoparticles C18 nanoparticles allow simple separa tions utilizing hydroph obic interactions; however, aptamer nanoparticles offer the possibi lity of selective extractions in complex samples through affinity inter actions of the analyte with immobilized aptamers. To demonstrate, magnetic nanoparticles were functionalized with DNA aptamers. With typical dissociation constants in the range of submicromolar to picomolar values, a growing number of aptamers are being described as scientific and biot echnological tools. The aptamer chosen here was selected for D-vasopressin and immobilized on the surface of the nanoparticles. While the aptamer offe rs selective analyte removal, nanoparticles function as the solid support for extraction. The magnetic property of the nanoparticles minimizes separation procedures, requiring on ly one step for extraction. Placing the particles directly into the liquid matrix al lows a rapid analysis procedure that limits analyte loss. As a result, lower abundance samples can be analyzed. The D-vasopressin aptamer offers high se lectivity, yet has a large dissociation constant (Kd = ~1M) allowing analyte removal from the particles.188 Aptamer functionality has been previously examined when attached to a solid support through avidin-biotin interactions.187 In this case, the aptamer show ed little to no deterioration of its binding abilities. To examine the selectivity of the aptamer once attached to the particle, two controls were conducted. First, the D-vas opressin functionalized magnetic particles were incubated with two dissim ilar peptides; angiotensin II (ASP-ARG-VALTYR-ILE-HIS-PRO-PHE) a nd bradykinin fragment 17 (ARG-PRO-PRO-GLY-PHESER-PRO). The aptamer did not have suffici ent nonspecific binding for either peptide to

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130 yield a mass spectrometry signa l after extraction. The s econd control for selectivity focused on whether the analyte would nonspeci fically bind to an aptamer functionalized particle. Individual aliquots of L and Dvasopressin were incubated with adenosine aptamer functionalized magnetic nanoparticles. The particle s failed to remove either analyte as shown by the lack of mass spectrometry ion signals. This control demonstrates a lack of nonspecific binding of vasopr essin analyte for a nonselective aptamer functionalized nanoparticle. The Kd values for the D-vasopressin aptame r with L and D-vasopressin are in a ratio of 1000:1.187 This ratio allows an interestin g control to exist for the aptamer functionalized particles. Ex traction of the L-vasopressin should present a limited mass spectrometry signal, demonstrating minimal ex traction efficiency. The D-vasopressin ion signal should larger due to a more effici ent extraction. Figure 5-10 shows the mass spectra for L and D-vasopressin before and after the aptamer func tionalized nanoparticle extractions. Figure 5-10A shows the L-vasopr essin molecular ion and sodium adduct. The bottom spectrum in Figure 5-10A s hows the nanoparticle extraction for Lvasopressin. Limited signal intensity was obtained due to the D-vasopressin aptamer’s limited affinity for L-vasopressin. Figure 5-10B s hows the molecular ion and sodium adduct for D-vasopressin. The bottom spectrum in Fi gure 5-10B shows the nanoparticle extraction for D-vasopressin. The ion signal counts for the two extractions are estimated at a ratio of ~1000:1 (D to L-vasopressi n), consistent with the Kd values for the aptamer of the two isomers. The D-vasopressin aptamer f unctionalized nanoparticles extract the Dvasopressin at binding values predicted. A dditionally, in Figure 5-10B the before (top)

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131 106010701080109011001110112011301140 m/ z Intensity 106010701080109011001110112011301140 m/ z Intensity 106010701080109011001110112011301140 m/zIntensity 106010701080109011001110112011301140 m/zIntensity B A Purchased D-Vasopressin with non-cyclic impurity Extracted D-Vasopressin without non-cyclic impurityH-Cys1-Tyr-Phe-Gln-Asn-Cys6-Pro-Arg-Gly-NH2 [M+Na]+ [M+H]+ 1085 1085 1087 1087 Figure 5-10. Mass spectra for L and D vas opressin before and after nanoparticle extractions are shown. Figure 5-10A shows the L-vasopressin before extraction (top) and after extraction ( bottom). The L-vasopressin was not extracted well using the D-vasopressin aptamer. Figure 5-10B shows the Dvasopressin before extraction (top) a nd after extraction (bottom). The Dvasopressin was extracted more efficien tly then the L-vasopressin. Also noticeable in the D-vasopressin is the lack of a mixture of disulfide and nondisulfide bond vasopressin. Only the disulfide bond containing D-vasopressin was extracted.

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132 and after (bottom) extraction spectra show the aptamer’s ability to extract only the disulfide bridge containing peptide. The original aptamer solution contained both the oxidized and reduced form of D-vasopressin; however, after extraction only the oxidized form was extracted by the aptamer nanoparticles. This hints to the aptamers ability to bind tertiary structure versus primary structur e. Also, for this particular aptamer, the disulfide formation is critical to the formation of an aptamer—D-vasopressin complex. The coupling of aptamer functionalized ma gnetic nanoparticle w ith AP liquid MALDI analysis offers a rapid analysis using hi ghly selective extraction agents and provides added sample clean-up. Conclusions The use of a liquid APMALDI matrix ha s allowed the development of a rapid analyte extraction technique. Using know n material types and functionalization chemistry, C18 and aptamer f unctionalized nanopart icles can act as scavenging agents for peptide molecules. Placing the particles directly into the liquid matrix allows a rapid analysis procedure that limits analyte loss and provides an improved preconcentration of the analyte. The C18 functi onalized silica particles allow extraction of a variety of peptides simultaneously while MS analysis can be done directly in the liquid matrix. The magnetic nanoparticles provided a si mplified separation procedure through the use of magnetic extraction. The particles also contained se lective functionalization for a more selective analyte-nanoparticle interac tion; permitting wider applications and the possibility of analyzing lower abundance sa mples. The aptamer nanoparticles allowed the selective extraction of reduced D-vasopres sin even in the presence of a structural analog. In addition, the extraction of Lvasopressin was possible, yet the extraction

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133 efficiency matched the 1000:1 dissociation c onstant ratio for D versus L-vasopressin using the D-vasopressin aptamer. These methods provide an avenue for advancement in liquid APMALDI sample preparation procedures. Optimizing the nanopa rticle synthesis and washing procedures will be useful for further experimentation. An evaluation of the particle capacity and extraction efficiency will also prove helpful for the possibility of quantitative studies. More importantly, with an increasing number of available aptamers and the growing interest in their applications, an array of particles for specific mass spectrometry analyses could result. While the m echanistic aspects of the MA LDI process are still unknown, applications involving the technique are thriving.

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134 CHAPTER 6 SECONDARY IONIZATION OF LASE R DESORBED NEUTRALS FROM ATMOSPHERIC PRESSURE MATRIX-ASSISTED LASER DESORPTION/IONIZATION Introduction Matrix-assisted laser deso rption/ionization (MALDI) produces ions from two convoluted steps—desorption and ionization. Determining the mechanisms of the individual steps is difficult because ion detec tion is affected by both processes as well as numerous instrumental parameters. Suppl ementary analysis methods, such as the fluorescence imaging of desorbed matrix pa rticles, adds valuable information by characterizing one process at a time. By in corporating other supplementary methods we may further assist in understan ding MALDI mechanistic events. Secondary ionization techniques used in conjunction with la ser desorption can provide a basis for examining the desorption pr ocess. By decoupli ng the ionization step, neutral populations can be independently stud ied. A variety of secondary ionization techniques (e.g., electron and chemical ionization) ha ve been used to determine neutral populations for vacuum laser desorption.198-199 The studies revealed that neutral molecules were desorbed ove r longer times and in larger quantities than the ion populations. Vacuum MALDI has similar ne utral molecule desorption properties. MALDI analysis yields ~104 neutral molecules for every one ion produced.200-201 Only a fraction of the total species present is then sampled by the mass spectrometer. Previously, the Harrison research gr oup introduced a s econdary ionization technique for IR laser desorption.202-203 In the technique, a co rona discharge provided

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135 reagent ions for the interaction with laser desorbed neutrals. This source has been referred as laser desorption atmospheric pre ssure chemical ionization (LD-APCI) for its combination of the two previ ously demonstrated techniques. Reagent ions from the corona discharge participate in chemical ionization reactions with neutral molecules produced during IR laser desorption. This chapter focuses on applying the techni que of atmospheric pressure chemical ionization with a corona di scharge to a UV APMALDI source. The laser desorption process can be studied indepe ndently, allowing the corona discharge to probe neutral molecule formation at fluence thresholds The key differences between the UV LDAPCI and the IR LD-APCI arrangements ar e the laser wavelength, mass analyzer, and AP inlet. Each of the above variations ha s played a significant role in how the more common UV MALDI laser, nitrog en 337 nm, has been applied to LD-APCI. IR lasers provide a larger population of neutrals and clusters, thus forming fewer ions.204 The UV laser yields a reduced neutra l/cluster population compared to the increased ion fractions from IR laser desorption. In previous IR LD -APCI experiments, an ion trap was utilized, which provided ion accumulation and temporal sampling. On the contrary, an oaTOFMS, which operates asynchronously with th e laser pulse, prevented ion accumulation from multiple laser pulses. Atmospheric pressure inlets dictate both arrangement of source potential fields and type of declustering method applied. Previously, declustering was done using a heated capillary inlet. Th e inlet extended out from the spectrometer. However, with the oa-TOFMS, cluster preventio n is controlled by a nitrogen current gas, which directs large gas flows towards the sa mple. While each method provides benefits,

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136 the source potential fields in the counter-cu rrent design are more space restricted, which is problematic for ion co llection and transmission. Experimental Method Atmospheric Pressure MALDI oa-TOFMS The atmospheric pressure MALDI source a nd the oa-TOFMS have been described in Chapters 2 and 3. The atmospheric pres sure interface uses a h eated counter-current nitrogen gas (100C, 5 Lmin-1) for cluster prevention. The ion transport field between the target and nozzle was 1100 Vmm-1. Gas pressures in the skimmer and RF-only quadrupole were 3 Torr and 10-1 Torr respectively. As noted in Chapter 2, the laser source was a 337 nm nitrogen laser (VSL337-ND-S, Spectra-Physics, Mountain View, CA, USA). Pulse energies ranged from ~5-180 J, measured directly using a pyroelectric detector (J4-09-030, Molectron Detector, Inc., Santa Clara, CA, USA). Variations in the energy transmitted to the sample originated from a gradient UV attenuating wheel (Reynard Corporation, San Cl emente, CA, USA) and an adjustable iris (Edmund Optics Inc., Barrington, NJ, USA). Target positio ning was accomplished using a piezoelectric transducer driven xyz stage (8302/IPico Driver, New Focus, San Jose, CA, USA). Mass spectra shown in this chapter are an accu mulation of summed spectra for 1-5 minutes. Corona Discharge The corona discharge was positioned using a flexible mount (flex lock 9940, New Focus, San Jose, CA, USA) and was attached to the mount with insulating connectors (Thorlabs, Newton, NJ, USA). High voltage, 0-12 kV, was applied using an external power supply (225-20, Bertan, Hicksville, NY, USA). Current was monitored directly on the power supply module. The needle distance was adjusted using a motorized xyz stage

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137 (CMA-12CCCL/ESP300; Newport, Irvine, CA USA). The optimal position for the needle was on-axis with orifi ce and 10 mm away. Relative to the target, the needle was ~8 mm back and 2 mm above. A sample execu table file, Object 6-1, of the LD-APCI arrangement demonstrates the configuration. The executable file was produced using a computer aided drawing program (Solid Work s, Concord, MA, USA) and shows a scaled view of the target, corona needle, and AP interface. Object 6-1. A file showing the orientati on of the LD-APCI source (1.8mb, LDAPCI.exe, repeating play file). Figure 6-1 is a diagram of the laser de sorption atmospheric pressure chemical ionization source. Figure 6-2 displa ys a photograph of the LD-APCI source. e-m* N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2+ + + + + eeeO2 O2 O2 O2 N2 N2N2+ N2+ O2+ N4+ N4 e-m* N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2+ + + + + eeeO2 O2 O2 O2 N2 N2N2+ N2+ O2+ N4+ N4 Target Skimmer Corona 2 kV400 V65 V 6 kV e-Oxygen Nitrogen Electron N2 O2L aser Figure 6-1. A diagram of the laser desorpti on atmospheric pressure chemical ionization source is shown. The orientation of the corona needle, target, laser, and interface can be seen.

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138 XYZ-translation stage Nitrogen Laser Focusing lens Target assembly Corona Assembly Figure 6-2. A photograph of the LD-APCI source is shown. The corona needle has been added to an atmospheric pressure matrix-assisted laser desorption/ionization source. The corona needle assembly is positioned by an xyz translational stage and held in place using a flexible lock mount. Matrix and Analytes Liquid matrices were prepared by mixing -cyano-4-hydroxycinnamic acid (CHCA) with a liquid support. The liqui d support was equal parts of 50% aqueous ethanol and diethanolamine (DEA). Chromophore concentr ation, CHCA, was 600 mM. To ensure homogeneity and dissolution, th e matrix solution was sonicated for 10-15 minutes. Immediately before analysis the ma trix was vortexed for 10 seconds. For each

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139 analysis, 0.5 L analyte solution and 0.5 L matrix solution were deposited on target, and in the order presented. For direct analysis, analytes were prepared in aqueous 0.1% TFA. Stock solutions of analyte were prepared at 50 pmolL-1, and diluted as noted. All analytes (angiotesin II, bradykinin fragment 1-7, bradykinin, rese rpine, and spiperone), matrix, and solvents were used unpurified (Sigma Al drich, St. Louis, MO, USA). Results and Discussion Secondary Ionization of Desorbed Neutrals Initial experiments demonstrated reagent ions interacting with UV laser desorbed neutral molecules, producing protonated mol ecular ions. The source was operated with the corona discharge powered on and off wh ile using APMALDI desorption for neutral formation. Figure 6-3 shows the total ion chromatogram as the source is operated in three different modes: LD-APCI, corona only, and APMALDI. In all three source modes, the target wa s maintained at the same position and only the corona voltage and/or the laser were turned on or off. Increased ion signal intensity is present with the corona discharge added to UV laser desorption. The reagent ions formed from the atmospheric pressure discharge allo wed ionization of MALD I neutrals. In the APMALDI and LD-APCI modes, the laser power was attenuated providing minimal laser fluence. The lower fluence reduced the la ser desorption ion signals allowing increases from reagent ions to be viewed. Redu ced fluence was obtained by limiting the pulse energy with a neutral density filter wheel and th en altering the diameter of a variable iris placed after the laser beam out put. With the iris diameter at ~3 mm, pulse energy was reduced to ~8 J.

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140 0 5 10 15 20 25 30 35 40 020040060080010001200 Time (s)IntensityN H N O O C H3O C H3O CH3O O O CH3O O CH3C H3 Reserpine + + + + + + APMALDI* LD-APCI Corona Figure 6-3. The total ion chromatogram for three modes of source operation is shown. Total ion counts were monitored for LD -APCI, corona only, and low fluence APMALDI modes. The laser spot size was altered to ~2 00 m yielding a fluence of ~250 Jm-2. Therefore, with the corona needle off, the source s hould be considered attenuated APMALDI. A comparison of the optimal APMALDI conditi ons versus LD-APCI shows much larger analyte ion intensity for APMALDI. Ma ss spectra demonstrating the APMALDI and LD-APCI modes are shown in Figures 6-4A and 6-4B, respectively. A weak APMALDI ion signal, due to the at tenuated laser flux, could be enhanced when the corona discharge was operated. Wh ile numerous differences were present for UV LD-APCI versus IR LD-APCI, results show that ion molecule reactions still occur.

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141 0 5 10 15 20 25 30 35 40 45 50 4005006007008009001000 m/zIntensity + + + LD-APCI LD-APCI[M+H]+ 0 5 10 15 20 25 30 35 40 45 50 4005006007008009001000 m/zIntensity + + + APMALDI APMALDI *[M+H]+ A B Figure 6-4. Mass spectra for reserpine using a CHCA liquid matrix in A) APMALDI and B) LDAPCI source modes are shown. Each spectrum was a 1 minute summation. The mass spectra correspond to the ion chromatogram in Figure 6-3.

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142 Corona Discharge The two-step approach used in LD-APC I (laser desorption and then chemical ionization) affords additional analytical utility but requires individua l optimization. Both the laser desorption and the corona discharge pr ocesses must be characterized to evaluate the technique. For the corona discharge, th e needle voltage plays a vital role. Evaluating needle potentials has shown that the discharge can tr ansition from a corona discharge to glow discharge as voltage is increased. Additiona lly, during the process, ion signal intensities drop. Discharge mode The discharge mode affects LD-APCI signa l intensities. The corona discharge mode allows ion signal increas es; however, when the discharge moves to the glow mode, degradation of ion signals occurs. Townsend’ s semi-empirical relationship for a corona discharge provides a means to di stinguish discharge transitions.204-205 Equation 6-1 presents the Townsend semi-empirical relationship: 0() I kUUU (6-1) where I is corona current, U is potential difference across electrodes, U0 is the onset voltage of the corona discharge (a func tion of corona needle geometry), and k is a factor that is inversely proportiona l to the gas density and interelectrode distance. A linear relationship holds for a corona discha rge, but not for a glow discharge. As the relationship turns to exponential increase, th e system moves from a corona to a glow. By plotting I/U versus U the transition to a glow di scharge can be determined.

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143 Figure 6-5 shows the transition from a corona to glow discharge occurring at 9 kV with 16 A of current. 0 0.5 1 1.5 2 2.5 024681012 Needle Voltage (kV)Current / Voltage (A / kV)0 10 20 30 40 50 60Intensity Figure 6-5. The figure shows the transition fr om a corona to glow discharge. The intensity for the angiotension II molecular ion was tracked during this transition. The molecular ion reaches a maximum before the transitions occurs. Needle distance from the orifice is ~8 mm. The intensity for the angiotensin II molecular ion was tracked during this transi tion. The molecular ion reaches a maximum before the transition occurs. Water clusters The reagent ions created from the corona discharge at atmospheric pressure have been studied numerous times.206-208 Reagent ion populations for atmospheric pressure

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144 chemical ionization are primarily solvated protons.12 The steps for positive reagent ion formation are: 22422 NNNN (6-2)4222NHOHON (6-3)223HOHOHOOH (6-4) followed by water clustering. The dominant positive reactive ions are H+(H2O)n, the value of n depending on the partial pressure of wa ter in the air. Providing the proton affinity for the analyte is greater than that of the reagent ion, a proton transfer reaction will occur. The proton affinity for monosolvated water is 166.5 kcal mol-1; therefore, analytes with a higher proton affinity will be protonated. Most nitrogen containing species exhibit proton affinities that range from 205 to 240 kcal mol-1. Thus, peptides and small molecule pharmaceuticals present an avenue for a proton transfer reactions. When the desorption target was removed a nd the heated counter-current gas flow turned off (reducing cluster prevention), reag ent ions produced from the corona discharge could be sampled. Figure 6-6 shows a mass spectrum collected with the corona needle placed ~10 mm from the inlet orif ice. The voltage was set at 6 kV with the current near 13.5 A. The peaks detected between m/z 50 200 represent a series of water clusters. This data verifies that reagent ions are being formed when the corona discharge is activated at ambient conditions.

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145 0 250 500 750 1000 050100150200m/ z Intensity[2(H2O)+H]+[3(H2O)+H]+[4(H2O)+H]+[8(H2O)+H]+[9(H2O)+H]+[10(H2O)+H]+[11(H2O)+H]+[5(H2O)+H]+[6(H2O)+H]+[7(H2O)+H]+ Figure 6-6. A mass spectrum of the water cluste rs produced from the corona discharge in air is shown. The corona needle had a ~50 m radius at the tip, and was in a point-to-plane configuration with the inlet orifice. The distance from tip to inlet was 10 mm. App lied voltage was 6 kV. Neutral Molecule Fluence Threshold Reducing the laser fluence below the ion threshold fluence for APMALDI yields no ions during the laser desorption step. Fo r LD-APCI, ion detection resulted from ionmolecule reactions with the neutral popul ation. Probing the neutrals created at atmospheric pressure was possible by using the reagent ions formed from the discharge. Figure 6-7 shows the voltage dependence of an analyte ion signal, bradykinin 1-7, when using a corona discharge with lo w fluence laser desorption (~100 Jm-2).

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146 0 1 23 4 5 6 7 8 9 1002468Voltage (kV)Current(A)0 10 20 30 40 50Molecular Ion CountsBradykinin Fragment 1-7 Figure 6-7. A plot of the anal yte ion intensity and corona current as the needle voltage was adjusted with low fluence laser de sorption. The analyte is bradykinin fragment 1-7. The spectra confirmed that neutral molecu le populations exist below ion threshold levels. As the voltage was increased, the cu rrent shows that reagent ions begin to be produced. Once the current was near 4 A th e analyte ion signals increased through ionmolecule reactions. At a corona voltage of zero, ion production is from laser desorption only, so ions were produced. Ions ar e detected only with the corona on. Figures 6-8, 6-9, and 6-10 demonstrate ne utral molecule probing using the corona discharge as a secondary ioniza tion source. Keeping the flue nce just below ion threshold values allows for ion-molecule reactions fr om the water cluster reagent ions to produce protonated molecular ions. The fluence wa s maintained throughout the experiments.

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147 0 50 100 150 200 250 300 350 400 450 500650700750800850900m/zIntensity Bradykinin Fragment 1-7 + + + LD-APCI LD-APCI + + + APMALDI APMALDI *[M+H]+[M+Na]+[M+H+DEA]+ Figure 6-8. Mass spectra of bradykinin frag ment 1-7 using low fluence APMALDI and LD-APCI modes are shown. The co rona discharge enables protonated molecular ions to be produced for br adykinin fragment 1-7; 25 picomoles were placed on target. Each spectrum is a 5 minute sum. The corona discharge, acting as a secondary ionization technique, did not result in enhanced UV APMALDI signals. The produc tion of ions was only seen during lower fluence studies. Figure 6-11 shows the produc tion of protonated molecular ions using higher fluence values (~600 Jm-2); pulse energy of 60 J a nd a laser spot size of ~300 m were used with no iris attenuation. With the larger population of neutrals pr esent during desorption the stagnation or reduction of ion signals with increasing co rona voltage was puzzling. To further investigate the negative effect s of the corona discharge, de tails of the ion transmission were sought.

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148 0 50 100 150 200 250 300 10001100120013001400150016001700180019002000 m/zIntensity P14R Peptide + + + LD-APCI LD-APCI + + + APMALDI APMALDI *[M+H]+ Figure 6-9. Mass spectra for P14R using low fluence APMALDI and LD-APCI modes is shown. The corona discharge enable s protonated molecular ions to be produced for P14R; 25 picomoles were placed on ta rget. Each spectrum is a 5 minute sum. 0 10 20 30 40 50 60 70 80 90 100 20002100220023002400250026002700280029003000 m/zIntensity ACTH Fragment 18-39 + + + LD-APCI LD-APCI + + + APMALDI APMALDI *[M+H]+ Figure 6-10. Mass spectra fo r ACTH fragment 18-39 usi ng low fluence APMALDI and LD-APCI modes is shown. The co rona discharge enables protonated molecular ions to be produced for AC TH fragment 18-39; 25 picomoles were placed on target. Each spectrum is a 5 minute sum.

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149 0 1 2 3 4 5 6 7 8 9 10 02468Voltage (kV)Current (A)0 500 1000 1500 2000 2500Molecular Ion CountsBradykinin Fragment 1-7 Figure 6-11. A plot of the analyte ion intensity and corona current as the needle voltage was adjusted with high fluence laser de sorption. The analyte is bradykinin fragment 1-7. Ion Transmission Potentials over 8 kV on the corona needle resulted in reductions in the analyte ion signals. This can be attribut ed to ion collection problems fr om the high potential fields created at the needle point. Increased vo ltages altered the potential fields, which are required for effectively transferring ions into the mass spectrometer. The corona acts as an intense point source. For both APMALD I and LD-APCI, potential fields for ion transport were critical. Additional evidence of the corona discharge disturbance was seen with the major deviations in signal from needle positioning. At ~2 mm above the target and 10 mm from the orifice the system operate d well. Movements beyond 0.5 mm in any direction resulted in no ions detected.

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150 Ion collection problems may also be a factor for the diminished LD-APCI signals at higher laser fluence. The ions present for APMALDI create a region of higher space charge preventing corona reagent ions from entering the orifice. Ion transmission was approximated using an ion optics computer modeling software program (SIMION 3D 7.0, Scientific Instrument Services, Ringoe s, NJ, USA). Dr. Tony Appelhans, Idaho National Laboratory, compar ed SIMION models for both the APMALDI and LD-APCI arrangements. It was determined that at higher voltages, the potential fields needed to transport ions into the mass spectrometer orif ice were disturbed. Only operating at lower voltages, or with a different source arra ngement, could an enhancement of APMALDI signals occur. Conclusions The construction of a UV LD-APCI s ource and its characterization was demonstrated in this chapter. Atmospheric pressure chemical ionization, an established ion production technique, allowed secondary io n-molecule reactions to probe the neutral molecules produced from UV laser desorption by attenuating the laser. The addition of secondary ionization processes may prove usef ul not only as an IR MALDI enhancement technique, but also as a methodology to probe atmospheric pressure UV desorbed neutral populations.

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151 CHAPTER 7 CONCLUDING REMARKS Research in genomics, proteomics, and systems biology has necessitated improvements in biomolecule analysis. In particular, the techni que of biological mass spectrometry has received well-deserved attention for its ro le in biopolymer characterization. The expansion of biological mass spectrometry can la rgely be attributed to the development and characterization of two complementary ionization sources, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Our research contributes in this area by expanding upon atmospheric pressure ionization techniques, in particular MALDI, and th e unique applications available to them. For MALDI analysis a suitable matrix for energy absorption and transfer to the analyte is required. Recent advances allo w atmospheric pressure MALDI sources to be coupled to a variety of mass analyzers. Th e ambient conditions available extend matrix formulations from solid crystalline format s to homogeneous liquid sampling systems. Ultraviolet compatible liquid matrices have not been previously studied at AP, and allow possible variations focused on desorption a nd ionization. The liquid matrices we examined provided self-renewing surfaces that eliminated sampling heterogeneity, increased sample lifetime, and offered shot-t o-shot reproducibility. Liquid sampling also allows measurements for laser penetrati on depth, solution viscosity, and chromophore concentration. Together these fundament al studies contribute to the fundamental knowledge of MALDI.

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152 With formulation studies of the liquid matrices completed, the focus shifted to characterizing the desorption and ionization pro cesses inherent for liquid systems. While the details of ion formation in MALDI are still heavily debated, parametric studies relating laser variables to analyte signals must be conducted for application development. The measurements made throughout this di ssertation provide a basis for further atmospheric pressure liquid matrix applications. Variations in laser parameters allowed for the characterization of the laser-liquid interface. Optimized fluence is both matrix and pressure dependent; therefore, fluence levels were adjusted while monitoring analyte ion yields. The liquid matrix at AP tolerated higher fluences while providi ng protonated molecular ions and minimal fragmentation. At the highest pulse energies, frag mentation and particle ejections played a role in decreasing analyte ion intensities. At higher pulse ener gies, nanometer sized particles were ejected in conj unction with a decay in analyt e ions. Ion yields can be related directly to laser parameters, yet MA LDI plume interactions also played a large role in their intensities, as demonstrat ed through analyte signa l suppression studies. Additionally, the examination of liquid versus solid matrices suggests the possibility of softer ionization. Liquids pr ovide added absorption of laser energy without increased analyte fragmentation. A soft er ionization process and reproducible longlasting ion signals may allow the liquid matrix to be used in high-throughput automation analyses. The liquid matrix offers advantag es that complement current MALDI methods. Liquid sampling reduces sample preparation, compared to solid matrices; however, during biological analysis se parations are often the rate determining step. We have explored further reductions in preparati on time for biomolecules with the use of

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153 nanoparticles as a rapid analyte extraction technique. Using known material types and functionalization chemistry, C18 and aptame r functionalized nanopa rticles can act as scavenging agents for peptide molecules. Pl acing the particles directly into the liquid matrix allows a rapid analysis procedure that limits analyte loss and provides an improved preconcentration of the analyte. Th e C18 functionalized silica particles allows extraction of a variety of peptides simulta neously while mass spectrometry analysis can be done directly in the liquid matrix. F unctionalized nanoparticle s provided specific extraction, retention, and concentrati on of simple biopolymers. The aptamer nanoparticles allowed the sele ctive extraction of reduced D-vasopressin even in the presence of a structural analog. This appr oach introduces nanoparticles as an extraction and concentration mechanism for low abundance analytes in complex systems. These methods provide an avenue for advancemen t in liquid APMALDI sample preparation procedures. More importantly, with an incr easing number of available aptamers and the growing interest in their applications, an ar ray of particles for specific mass spectrometry analyses could result. While the mechanis tic aspects of the MALDI process are still unknown, applications involving the technique are thriving. While still extensively debated, the MALD I process involves some combination of desorption and ionization. During these pr ocesses, a large po pulation of neutral molecules is created in the MALDI plume. Stud ies to date have revealed that in MALDI, for every 104 molecules desorbed perhaps only 1 ion is produced. Supplemental ionization techniques can offer a clea r view of the majority species (i.e., neutral molecules) produced during MALDI. At atmospheric pressure conditions, experimentation on the available neutrals can be accomplished. Using reagent ions from

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154 an atmospheric pressure corona discharge we are able to probe the MALDI plume. We conducted experiments with a UV laser to study th e details of the ion-neutral interactions. Important in the addition of a secondary ioni zation is the population of reagent ions and the ion transfer parameters. Preliminary results have shown that UV laser desorbed neutrals do interact with atmospheric pressu re reagent ions; however, the individual optimization of each process is needed. We expect this technique to offer insight into several pressing questions in the MALDI mechanisms. The neutral molecules provide an avenue for enhancing limits of detection, ionizing non-ionized anal ytes, and probing ionmolecule chemistry. The construction of an APMALDI ionizati on source and its characterization using UV liquid matrices is shown. Ambient condi tions present at AP allow new opportunities for matrix development. The addition of secondary ionization processes may prove useful not only as an enhancement technique but also as a methodology to probe neutral populations at low laser fluence levels in APMALDI. Liquid matrices and other interesting applications are possible with a simple atmospheric pressure source. The exploitation of AP interfaces with atmospheric pressure la ser desorption techniques can provide needed advances in biological analysis. Further research into the us e of liquid matrices and thei r inherent applications may also demonstrate added analytical ut ility. Studies should embrace additional chromophores and other analyte classes. While the liquid matrix provides immediate benefits for APMALDI analysis with its ease of use, liquid matrix possibilities include an on-line liquid UV APMALDI ion source for ch romatography and reaction monitoring.

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155 Nanoparticle use with liquid APMALDI also presents interesti ng applications for bioanalytical analysis. Studies focusing on analyte-nanoparticle incubation ratios, functional group selectivity, and complex mixture extraction will provide further evidence for nanoparticle usage in biological mass spectrometry. Optimizing the nanoparticle synthesis and washing procedures w ill be useful for further experimentation. An evaluation of the particle capacity and extraction efficiency will also prove helpful for the possibility of quantitative studies. During the optimization of the APMALD I source, the focus was on increasing reproducibility and analytical utility; however, there are possi bilities for signal-to-noise enhancement. The MALDI source uses a laser pulse not synchronized to the TOF repeller pulse. At a laser repetition rate of ~20 Hz a semi-continuous ion stream is produced. Since the liquid matrix offers cont inuous sampling without laser repositioning, the use of a high repletion rate solid-state UV laser could provide signal increases. Higher laser frequencies yield more overlap for individual i on packets producing a denser ion beam. Alternatively, decreasing the laser fr equency may reduce chemical noise. By gating the acquisition for each laser pulse, back ground ions can be discriminated against. Preliminary results have shown individual ion packets present regions of maximum analyte signal and minimum chemical b ackground. The evaluation of temporal properties present for APMALDI could allow th e optimization of the source as well as provide benefits for seconda ry ionization additions. There are many aspects of the UV LD-APCI s ource that still must be considered. For the immediate future, a redesign of the source interface should assist in optimizing

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156 the potential fields for ion collection and tran smission. Our initial studies have shown that as the discharge transitions from a corona discharge to a glow discharge, ion signal intensity drops. At this time, it is not clea r whether the reagent ions produced in each discharge mode directly affect ionization efficiency or if field disturbances are the sole factor. Source parameters should be empirica lly and theoretically st udied using prototype sources and SIMION modeling. The inclusion of fluid dynamics calculations may assist in evaluating ion collection at atmospheric pressure. The development of the LD-APCI source also suggests the use of reagent gas st udies. A detailed understanding of reagent ion populations and interactions may allow se lective ion-molecule interactions in the APCI environment.

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157 APPENDIX MALDI MATRICES AND PREPARATION MALDI Matrices A variety of matrix-assisted laser desorp tion/ionization (MALDI) matrices are used on a regular basis. While not all matrices ar e routine, each seems to have its own unique properties: enhanced ionization for a chemi cal class, reduced background for a sample matrix, or eases preparation pr otocol for convenient use. Listed in Table A-1 are the more common MALDI matrices. Preparation Protocols MALDI preparation protocols are often as daunting to choose as the MALDI chromophore itself. Additional preparation pr otocols are being introduced — even this dissertation seems to suggest an advanced a pproach. While each preparation protocol provides benefits, there are some standard soli d matrix preparations that seems to work well for general analyses. However, the auth or is biased towards atmospheric pressure liquid preparations. Dried-Droplet This is the original sample prep aration procedure introduced in 1988 by Hillenkamp and Karas.3,36-37 While modifications in the process exist, the basic procedure is relatively simple. A portion of matrix solution is mixed with an analyte solution left to dry. The result is a solid deposit of analyte-doped matrix crystal. One benefit of this protocol method is that the analyte/matrix crystals may be washed to

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158 remove non-volatile components (i.e., interfering salts).42 This method tolerates some salts in mM concentrations and is a good c hoice for mixtures of proteins or peptides. Vacuum-Drying A slight variation on the dr ied-droplet method is the addition of vacuum drying for crystallization. In this method, the final anal yte/matrix drop applied to the target is dried in a vacuum chamber. Vacuum-drying can reduce the size of the analyte/matrix crystals, which may assist with crystal homogeneity.9-10 Fast-Evaporation The fast-evaporation method was introduced with the main goal of improving the resolution and mass accuracy of MALDI measurements.42 Matrix and sample are handled separately. First, the matrix solution is desposited onto the target and allowed to evaporate. Next, on top of the matrix, the analyte solution is depos ited and allowed to dry. The process delivers stable and long lived matrix films that can be used to precoat MALDI targets.10 Table A-1. Common MALDI matrices. The typical laser wavelength and chemical classes used are also listed; how ever, these are only guidelines not requirements. Matrix Mass (Da) Laser (nm) Analyte Class 3-Amino-4-hydroxybenzoic acid 153 337 Oligosaccharides 2,5-Dihydroxybenzoic acid (DHB) 154 266,337,355 Oligosaccharides, peptides, nucleotides, oligonucleotides 5-Hydroxy-2methoxybenzoic acid 168 337 Lipids 2[4hydroxyphenylazo]benzoic acid (HABA) 242 266,337 Proteins, lipids

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159 Table A-1. Continued. Cinnamic acid 148 337 General? -Cyano-4hydroxycinnamic acid (CHCA) 189 337,355 Peptides, lipids, nucleotides 4-Methoxycinnamic acid 178 266,337,355 Proteins Sinapinic acid 224 266,337,355 Lipids, peptides, proteins Ferlulic acid 194 266,337,355 Proteins 6,7-Dihydroxycoumarin (esculetin) 178 337 Lipids, peptides 3-Hydroxypicolinic acid (HPA) 139 337,355 Oligosaccharides Picolinic acid (PA) 123 266 Oligosaccharides 3-Aminopicolinic acid 138 266,337,355 Oligosaccharides 6-Aza-2-thiothymine (ATT) 143 266,337,355 Oligosaccharides, lipids 2,6-Dihydroxyacetophenone 152 337,355 Proteins, oligonucleotides 2,4,6Trihydroxyacetonphenone 168 337,355 Oligonucleotides Nicotinic acid 123 266,337,355 Proteins, oligonucleotides 1,5-Diaminonaphtalene 158 337 Lipids Succinic acid 118 2940 Oligosaccharides Urea 46 2940 Oligosaccharides Caffeic acid 180 337 Peptides, proteins Glycerol 92 2940, 10600 Peptides, proteins 4-Nitroaniline 138 337 Peptides, proteins

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172 BIOGRAPHICAL SKETCH Kevin Paul Turney was born in Lakenh eath, England. He attended Eastern Washington University in Ch eney, Washington, graduating summa cum laude with a Bachelor of Science degree in chemistry and a minor in physics. In the fall of 2000, he enrolled in the Analytical Division of the Chemistry Department at the University of Florida. Under the direction of Professor W. W. Harrison, he completed his graduate studies with a Doctor of Philosophy in December 2004.


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Physical Description: Mixed Material
Copyright Date: 2008

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Full Text












EXTENDING ATMOSPHERIC PRESSURE MASS SPECTROMETRY:
DESORPTION AND IONIZATION CONSIDERATIONS















By

KEVIN TURNEY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Kevin Turney




























This dissertation is dedicated to my parents, Marsha and James, for their support; to my
sister, Angela, for her friendship; and to my wife, Kimberly, for her patience.






Strange how much you've got to know before you know how little you know.
-unknown















ACKNOWLEDGMENTS

Any success that I have had is entirely because of the people who have mentored,

guided, and literally pushed me in the right directions. I could never show my full

gratitude, yet below is my brief and pathetic attempt.

I am indebted to my research advisor, Willard W. Harrison, for welcoming me into

his laboratory and the Harrison Group family. Through the years I have had a chance to

admire his scientific passion and managerial wit. The discussions in his office and at the

card table will always be remembered.

The greatest benefit as a Harrison group member has been the connection shared

with Dr. James D. Winefordner's Laboratory. As a research professor, Doc is supportive

and compassionate of every graduate student. Also, I am grateful to Dr. Ben Smith for

encouragement, and an occasional burger. It has been a delight to work alongside the

Winefordner group members, enjoying the traditions and the history of the lab. The JDW

laboratory is truly a special place.

Many members of the chemistry department have been both my colleagues and my

friends. Dr. Eric Oxley has continued to support the Harrison group even after his

departure. I am appreciative of his friendship and his strength in lifting motorcycles.

There were times during graduate school that only a talk with Dr. Paige Oxley could

brighten my day-I still miss the discussions. I thank Li Qian for teaching me science

and game strategy.









Some people always wear a smile, walking through the lab making everyone feel

just a little bit better. Over the last few years, for the Winefordner and Harrison groups,

that person has been Dr. Tiffany Correll. Her sense of humor and good-natured spirit are

truly unique. She always knew how to put me in my place-Go Dumplings.

Lock two people in a room for a few years and they may become friends or they

may become enemies. I am happy to say the years I spent with my lab mate were filled

with scientific discussion, data collection, and laugher. I consider Dr. Elizabeth Pierz

Hastings a cherished and respected friend.

Many thanks also go to Dr. Wiehong Tan's laboratory, the members provided me

with scientific instrumentation and camaraderie. Special thanks go to a good friend, Tim

Drake, for this help with science and university politics.

I may forget the science I learned along the way, but I will not forget the friends I

made. My most prized memories of graduate school are the late nights and the coffee

runs-both of which were not conducted alone.

My path to graduate school was long and not without ordeals. I would never have

gotten here without the help of an influential Professor, Dr. Suzanne Bell. She provided

an engaging scientific environment that made me want to be a part of science. I am

deeply indebted to her for both scientific and personal growth.

On a more personal note, I thank my parents, James and Marsha DeMotta, for their

never-ending support. I try my best everyday to simply make them proud. I thank my

sister, Angela, for always being there-in good times and bad. Special thanks also go to

my brother-in-law, Brian; and to my three wonderful nieces, Amanda, Alexis, and

Abigail.










To one person I am most indebted: my wife Kimberly. She may be last on paper,

but she is always first in my mind. I thank her for her support, patience, and love-more.

Neither this dissertation nor my graduate work would have existed without her.

If I've failed to iterate my thanks, or I left a crucial individual or two out, please do

not fault me-as you know, I need help from time to time.


"Piled Higher and Deeper" by Jorge Cham
www phdcomics com


or
I ;


'



i.
-F!

c
















TABLE OF CONTENTS

Page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ....................................................... ............ ....... ....... xi

L IST O F F IG U R E S .... ...... ................................................ .. .. ..... .............. xii

LIST O F O B JE C T S .... .................................................... .. ....... ............. xvi

ABSTRACT ............. .................................................................. xvii

CHAPTER

1 MOLECULAR MASS SPECTROMETRY ...........................................................1

H historical B ackgrou n d ........................................................................ .............. .
Ionization T echniques.......... ..... ............................................................ ........ ... ...
Electron Ionization ...................................... ........................ ....
C hem ical Ionization........... ........................................................ ................ .5
B iom olecule A naly sis............ ... ........................................................ ........ .... .8
Energy-Sudden Approach .................................. .....................................8
P lasm a D esorption .......... ..... ......................................................... ........ .... .9
Fast A tom B om bardm ent ................................................... ........ ............... 11
Laser D esorption Ionization .......................................... .......................... 13
Matrix-Assisted Laser Desorption/Ionization ............................................. 14
M e c h a n ism s ............................................................................................ 1 6
M atrix considerations .............................................................. ............... 2 1
Tim e-of-Flight M ass Spectrom etry ........................................ ........................ 22
H historical Perspective ........................................ ........ .... ........ .. .... ........ 22
T im e-of-flight T h eory .............................................................. .....................2 3
K inetic energy spreads ........................................ ........................... 25
S p atial sp read s ................................................................. ............... 2 6
D esorption Ionization Techniques.................................... ....................... 27
C o n clu sio n s..................................................... ................ 2 8









2 DESIGN OF AN ATMOSPHERIC PRESSURE MATRIX-ASSISTED LASER
DESORPTION/IONIZATION SOURCE FOR AN ORTHOGONAL-
ACCELERATION TIME-OF-FLIGHT MASS SPECTROMETER......................29

Introdu action .................................................. .................................... 29
Background ................. ........................ ........... ...............29
Orthogonal-Acceleration Time-of-Flight .................. .............. ............... 29
Atm ospheric Pressure Inlets ............................................. ............... 31
Atm ospheric Pressure M ALDI........................... .................. ................... 33
E xperim ental M ethods....................................................................... ...................35
M ass Spectrom eter ........................ .... ............ ................. ..... .... 35
Electrospray Configuration ............................................ ........................... 37
Atmospheric Pressure MALDI Configuration ......................................................39
Sam ple P reparation ........ ................................................................ .. .... ..... .. 4 1
Solid m atrix ............................................................................. 41
L iquid m atrix .................. ..................................... .. ........ .... 42
R results and D iscu ssion .............................. ........................ .. ...... .... ...... ...... 42
E lectrospray E v alu ation ............................................................ .....................42
APM ALDI Source .................. .......................... ........ ................. 43
P ro to ty p e I .............................................................4 3
P rototy p e II............................................................54
A adjusting Interface Param eters ........................................ ........ ............... 55
C o n c lu sio n s..................................................... ................ 6 1

3 LIQUID SUPPORTS FOR ULTRAVIOLET ATMOSPHERIC PRESSURE
MATRIX-AS SISTED LASER DESORPTION/IONIZATION.............................62

Introdu action .................................................. .................................... 62
E xperim mental M ethods......................................................... ............ ............... 65
Atm ospheric Pressure M ALDI Source..............................................................65
Solution Preparation ............. ........ ...... ........ .. ... ........... .... ... 65
R results and D iscu ssion .............................. ........................ .. ...... .... ...... ...... 66
L iq u id M atric e s ............................................................................................. 6 6
Chrom ophore Concentration ........................................ .......................... 66
Support Liquid Variations ...........................................................................72
Solids versus Liquid M atrices ........................................ ......... ............... 76
Quantitation ............... ......... .......................82
M mixture A analysis ................. .... ........................ .. .. ....... ................. 84
C o n c lu sio n s........................................................................................................... 8 6

4 LASER DESORPTION CONSIDERATIONS USING LIQUID MATRICES AT
ATM O SPHERIC PRESSURE ............................................ ........................... 87

In tro d u ctio n ........................................................................................8 7
B background .............................................................................................................89









E xperim mental M ethods..................................................................... .....................90
Atmospheric Pressure MALDI oa-TOFMS ................................... ...............90
Fluorescence M easurem ents................................................... ............... ... 91
M atrix and A nalytes .................................................. .............................. 91
R results and D discussion ....................... ...... .......... ............... .... ....... 92
Liquid M atrix H om ogeneity ......... ................. ........................... ...... ......... 92
L aser Frequency ....................... .................. ... .... ........ ......... 94
F luence D ependence.......... ........................................................... .. .... ..... .. 96
P article Ejection ..... ....... ................................. .....................100
Plum e Interactions ................................ ..... ........ ... .... .. ............ 106
C o n clu sio n s.................................................... ................ 10 7

5 FUNCTIONALIZED NANOPARTICLES FOR LIQUID APMALDI PEPTIDE
A N A L Y SIS ..................................................... ................. 109

In tro d u ctio n ..................................................................... ............... 10 9
Experim mental M methods ........................................................................ 113
M materials ...................................... ............................................. 1 13
Nanoparticle Synthesis .............. ................. ..... ............... 113
Silica C 18 functionalized nanoparticles .......................... ......... .........113
M agnetic aptam er nanoparticles .............. ............................ .................114
M atrix and Analyte Preparation .............................................. .................. 115
Instrum entation ............................................................... ... .......... 115
E xtraction Procedures.................................................................................. 116
R results and D discussion ..................................... ................ .......... .... 119
B background ...............1... ...................1...................9..........
N anoparticle Characterization ...................................................... ................ 119
Peptide Analysis by Liquid APMALDI with C18 Nanoparticles .....................122
Peptide Analysis by Liquid APMALDI with Aptamer Nanoparticles..............129
C o n clu sio n s.................................................... ................ 13 2

6 SECONDARY IONIZATION OF LASER DESORBED NEUTRALS FROM
ATMOSPHERIC PRESSURE MATRIX-ASSISTED LASER
DESORPTION /ION IZA TION ......... .. ............ .................. .....................134

Introdu action ................................................................................................. 134
E xperim mental M ethod .................................................................... ..................... 136
Atmospheric Pressure MALDI oa-TOFMS ................................. ...............136
C orona D ischarge .................................................................... ................ .. 136
M atrix and A nalytes ................................................ .............................. 138
R results and D discussion ....................... ... ........... ....................... 139
Secondary Ionization of Desorbed Neutrals................................................139
Corona Discharge .................. .......................... .... ..... ............... 142
D ischarge m ode .................. .............................. ............ ... 142
W ater clu sters ................................................................ ............... 143









N eutral M olecule Fluence Threshold ........................................ .................145
Ion T ran sm mission ......... ...... ........ .. ...... .. .................. .. .................. 14 9
C o n clu sio n s.................................................... ................ 15 0

7 CON CLU D IN G REM A R K S .......... ................. ...................................................151

APPENDIX MALDI MATRICES AND PREPARATION .......................................157

L IST O F R E F E R E N C E S ........................................................................ .................... 160

BIOGRAPH ICAL SKETCH .............................................................. ............... 172
















LIST OF TABLES


Table Page

1-1 Common reagent gases for chemical ionization techniques.. .................................7

1-2 Typical laser wavelengths, photon energies, and pulse widths used for MALDI....17

2-1 Mass spectrometer conditions used to acquire electrospray spectra......................38

2-2 Mass spectrometer conditions used to acquire APMALDI spectra. ........................40

4-1 Conditions used to acquire the emission spectrum of the CHCA liquid matrix. ...103

A -i Com m on M ALD I m atrices. ........................................................ .....................158
















LIST OF FIGURES


Figure Page

1-1 D iagram of an electron ionization source................................................................3

1-2 Plot of ion yields versus electron energy. ....................................... ............... 4

1-3 Diagram of a chemical ionization source. ........................................ ............... 6

1-4 Plot of rate constants for decomposition and vaporization versus T .....................10

1-5 Diagram of californium plasma desorption ionization source ...............................11

1-6 Diagram of a fast atom bombardment ionization source. ........................................13

1-7 Diagram of a matrix-assisted laser desorption/ionization source.............................16

1-8 Diagram demonstrating the principle theory in time-of-flight mass analysis. .......24

2-1 Diagram depicting the complementary nature of atmospheric pressure ionization
so u rc e s ........................................................................... 3 4

2-2 Diagram of the orthogonal-acceleration time-of-flight mass spectrometer used in
the studies presented is show n. ........................................ .......................... 35

2-3 A photograph of the electrospray tip used for mass spectrometer characterization.38

2-4 A diagram of the components in a typical atmospheric pressure matrix-assisted
laser desorption/ionization source. ........................................ ........................ 39

2-5 Plots showing the A) total ion count chromatogram and B) a mass spectrum for the
analysis of reserpine. ............... ..... ........ ....... .. ...... ............. .....44

2-6 Plots showing the A) total ion count chromatogram and B) a mass spectrum for the
analy sis of verapam il......... ............................................................ ......... .... 45

2-7 A photograph of the first constructed APMALDI source (Prototype I). .................47

2-8 A mass spectrum of reserpine analyzed in a solid DHB matrix.............................48

2-9 Plot of the total ion count chromatograms for solid and liquid matrices ...............49









2-10 A photograph of the altered target assembly for liquid matrix analysis. ................50

2-11 Mass spectrum of reserpine in a DHB liquid matrix taken using the modified target
assem bly .............................................................................5 1

2-12 A photograph of the source and target assembly with the curtain gas cover
re m o v e d .......................................................................... 5 2

2-13 Mass spectra obtained with the target placed at a distance of A) 1.5 mm from the
orifice and B) 2 mm from the orifice. ........................................... ............... 53

2-14 A photograph of the second APMALDI source (Prototype II). .............................54

2-15 Three mass spectra taken using gas flow rates of A) 0 L-min 1, B) 1 L-min-1, and C)
5 L -m in -1 .............................................................................5 6

2-16 An illustration of the mass spectrometer interface................................................ 57

2-17 A three dimensional plot showing the relationship of target and nozzle voltage to
analyte ion yields .......................... ................. ......... 58

2-18 A three dimensional plot showing the relationship of nozzle and skimmer voltages
to analyte ion signal intensity ......... ............................................... ............... 59

3-1 The plot shows analyte ion and matrix background intensity as a function of CHCA
concentration in the liquid m atrix. ........................................ ....................... 68

3-2 Mass spectrum of five picomoles of angiotensin II analyzed using an optimized
CH C A liquid m atrix. ...................... .................. .............................70

3-3 An illustration of the UV-Vis absorption spectra collected for common MALDI
m atrice s. .......................................................... ................ 7 1

3-4 A plot of analyte ion intensity as a function of DHB concentration......................72

3-5 A chart of analyte intensity versus the percentage of DEA in the liquid matrix......74

3-6 A chart of analyte intensity versus solvent liquid used in the liquid matrix............75

3-7 Mass spectra of bradykinin fragment 1-7 comparing solid and liquid matrix
prep aration s. ....................................................... ................. 77

3-8 Mass spectra of angiotensin I comparing solid and liquid matrix preparations.......78

3-9 Mass spectra of ACTH fragment 18-39 comparing solid and liquid matrix
prep aration s. ....................................................... ................. 80

3-10 Mass spectra of P14R comparing solid and liquid matrix preparations...................81









3-11 A calibration curve for angiotensin II. ........................................ ............... 83

3-12 A calibration curve for bradykinin fragment 1-7. ....................................................84

3-13 A mass spectrum for a peptide mixture using the CHCA liquid matrix ................85

4-1 A plot showing individual ion packets resulting from liquid matrix analysis. ........93

4-2 A plot of analyte ion signals and pulse energy as a function of laser frequency. ....95

4-3 A plot of small molecule analyte ion yields as a function of pulse energy..............97

4-4 A plot of peptide analyte ion yields as a function of pulse energy. .........................98

4-5 The calibration of a UV neutral density filter wheel for pulse energy...................99

4-6 Mass spectra for reserpine illustrating fragmentation produced at higher pulse
energies (i.e., 10, 90, and 180 J). ...........................................................100

4-7 Mass spectra for bradykinin fragment 1-7 illustrating fragmentation produced at
higher pulse energies (i.e., 10, 90, and 180 pJ) .............................. ................. 101

4-8 Emission spectrum of the CHCA liquid matrix using 337 nm excitation ............102

4-9 Fluorescence image of the laser impinging on the liquid matrix surface.............104

4-10 Fluorescence image of particles ejected from the liquid matrix. .........................105

4-11 Mass spectra demonstrating analyte signal suppression. .......................................107

5-1 An illustration of the centrifugation technique used for nanoparticle extractions. 116

5-2 A diagram illustrating the utility of the nanoparticles with the liquid matrix........ 117

5-3 An illustration of the magnetic separation technique used for nanoparticle
extractions. ...................................................................... ..........118

5-4 Nanoparticle characterization using fluorescence analysis ..............................120

5-5 A chart of the protonated molecular ion mass spectrometry signals for three
p ep tid es ...................................... ...................................................12 3

5-6 Mass spectra collected from nanoparticle extractions ................. .. ...................124

5-7 A chart showing the effect multiple washing steps has on the sodium adduct signals
during m ass spectrom etry analysis.................................... ......................... 125

5-8 Mass spectra collected for a 1 [iM angiotensin II solution before and after
nanoparticle extraction. .............................................. ...................................... 126









5-9 A chart of the protonated molecular ion mass spectrometry signals for three varied
len gth p eptid es............................................................................... ............... 12 8

5-10 Mass spectra for L and D vasopressin before and after nanoparticle extractions. .131

6-1 A diagram of the laser desorption atmospheric pressure chemical ionization source
is sh ow n ............. ......... .. .............. ....................................................... 13 7

6-2 A photograph of the LD-APCI source. ............................................. ............ 138

6-3 The total ion chromatogram for three modes of source operation .......................140

6-4 Mass spectra for reserpine using a CHCA liquid matrix in A) APMALDI and B)
LD APCI source m odes ........................... .................................... ............... 141

6-5 A figure showing the transition from a corona discharge to glow discharge.........143

6-6 A mass spectrum for water clusters produced from the corona discharge in air....145

6-7 A plot of the analyte ion intensity and corona current as the needle voltage is
adjusted (low fluence laser desorption) ............................................................. 146

6-8 Mass spectra of bradykinin fragment 1-7 using low fluence APMALDI and LD-
A P C I m odes. ...................................................................... 147

6-9 Mass spectra for P14R using low fluence APMALDI and LD-APCI modes. ........148

6-10 Mass spectra for ACTH fragment 18-39 using low fluence APMALDI and LD-
A P C I m odes. ...................................................................... 148

6-11 A plot of the analyte ion intensity and corona current as the needle voltage is
adjusted (high fluence laser desorption)....................... ..................... 149
















LIST OF OBJECTS


Object Page

2-1 A file showing the ion source configuration after the target alteration for liquid
matrix analysis (1.8 mb, PrototypeI.exe, repeating play file). ................................48

2-2 A file showing the orientation of the prototype II source (1.8 mb, PrototypeII.exe,
playable file)..................................................................... ..........54

4-1 A video of the laser irradiating the liquid sample surface. (1.3 mb,
Liquidregeneration.mpg, 50 seconds). ........................................ ............... 94

4-2 A video of the laser irradiating the liquid sample demonstrating particle ejection at
110 0J pulse energy. (1 mb, 11Opulseenergy.mpg, 10 seconds) .........................105

4-3 A video of the laser irradiating the liquid sample demonstrating particle ejection at
140 0J pulse energy. (1 mb, 140pulseenergy.mpg, 10 seconds)...............................105

4-4 A video of the laser irradiating the liquid sample demonstrating particle ejection at
180 0J pulse energy. (1 mb, 180pulseenergy.mpg, 10 seconds)...............................105

4-5 A video of the laser irradiating the liquid sample demonstrating particle ejection at
180 0J pulse energy-magnified view. (1 mb, 11 pulseenergyzoomed.mpg, 10
se c o n d s) .................................... ............... ................. ................ 1 0 5

4-6 A video of the laser irradiating the liquid sample demonstrating particle ejection at
180 0J pulse energy-slow motion (non-false color) view. (5.5 mb,
180pulseenergyslowed.mpg, 50 seconds) ................................. ......................105

6-1 A file showing the orientation of the LD-APCI source (1.8mb, LDAPCI.exe,
repeating play file). ........................ .... ................ .. .... .... ............... 137















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EXTENDING ATMOSPHERIC PRESSURE MASS SPECTROMETRY:
DESORPTION AND IONIZATION CONSIDERATIONS

By

Kevin Turney

December 2004

Chair: Willard W. Harrison
Major Department: Chemistry

Biological mass spectrometry has received well-deserved attention for its role in

biopolymer characterization. Matrix-assisted laser desorption/ionization (MALDI) is one

method that allows the ionization of large intact biomolecules. For analysis, MALDI

requires a suitable matrix for energy absorption and transfer to the analyte. The most

widely accepted form of matrix is some form of solid that acts as an analyte host. While

compatible with the low pressure environment of a typical ion source, the matrix presents

a heterogeneous sample surface. Recent advances have allowed MALDI to be conducted

at atmospheric pressure (AP), extending its flexibility in source design and applications.

This research contributes in this area by expanding upon atmospheric pressure ionization

techniques and their unique applications.

To further sample analysis opportunities at atmospheric pressure, a liquid matrix

for UV APMALDI analysis was developed. Liquid matrices allow possible formulations

focused on desorption and ionization versus vacuum stability and source contamination.









The liquid matrices we examined provide a self-renewing surface that eliminates

sampling heterogeneity, increases sample lifetime, and provides shot-to-shot

reproducibility. Ambient condition liquid sampling also allows measurements for laser

penetration depth, fluence ranges, particle ejections, and chromophore concentration,

which can add to the study of MALDI mechanistic pathways. The liquid matrix offers

advantages that complement current MALDI methods.

Liquid sampling reduces sample preparation, compared to solid matrices; however,

during biological analysis separations are often the rate determining step. We have

explored further reductions in preparation time for biomolecules with the use of

nanoparticles. Functionalized nanoparticles provided specific extraction, retention, and

concentration of simple biopolymers.

During desorption process in MALDI, a large population of neutral molecules is

created. A secondary ionization technique, such as atmospheric pressure chemical

ionization, can provide reagent ions for interaction with laser desorbed neutrals. Results

show that UV laser desorbed neutrals do interact with atmospheric pressure reagent ions;

however, the individual optimization of each process is needed. Secondary ionization of

the neutral molecules provides an avenue for probing ion-molecule chemistry.

The exploitation of AP interfaces with atmospheric pressure laser desorption

techniques can provide needed advances in biological analysis.


xviii














CHAPTER 1
MOLECULAR MASS SPECTROMETRY

"Molecules having the same mass numbers but differing in weight by an amount
determined only by the difference in binding energies of the nuclear particles can
be clearly resolved.... Extension of the use of the instrument to the resolution of
heavy hydrocarbons should prove fruitful."
-Alfred O. Nier 19551


Historical Background

The origins of mass spectrometry are deeply rooted in the field of atomic physics.

Beginning with John Dalton, and his proposed new atomic theory, the idea that a unique

measurable property (relative atomic weight) existed for each element, was born.2 These

masses make the basis of measurement in "mass" spectrometry. Years after the atomic

theory was developed, J. J. Thompson was able to advance instrumentation and obtain the

first charge-to-mass ratios, e/m, for hydrogen and oxygen.3 The instrumentation directed

discoveries in both experimental atomic physics and mass spectrometry. This type of

paradigm, instrumentation progress leading to application-driven discoveries, has

followed the mass spectrometry field throughout its existence. Even today resolution and

precision advances have challenged ion formation mechanistic theories.4

Ionization Techniques

All mass spectrometers comprise five major components: sample inlet, ionization

source, mass analyzer, ion detector, and data acquisition system. At times, regions, such

as the inlet and the ionization source, can be combined (i.e., atmospheric pressure sources

(Vida infra)). However, even when regions may seem indistinguishable, each is required









for mass analysis. With mass spectrometry's basis in physics, it is not surprising that the

beginning dealt with ionization fundamentals. Using a combination of electrostatic and

magnetic fields for spatial dispersion and photographic plates for detection, physicists

focused on the ion source for further advancements.5 For instance, Thompson's initial

studies using mass spectrometry involved Goldstein's Kanalstrahlen, a glow discharge

-2
ionization technique.

While early sources provided easy access to gases, they did not provide convenient

analysis of solid samples. At least not until Arthur Dempster made use of a high-

frequency spark discharge to determine the isotopic analysis of platinum, palladium,

gold, and iridium.2 Ions were created in the energetic discharge by using conducting

samples as the counter electrode, but it was difficult to analyze semiconductors and

insulators. Typical of ionization techniques, advances in the field provide new avenues

for analysis (i.e., solid samples); the advancements led to additional fundamental

questions. The next step in ion source development eventually shifted the focus of mass

spectrometry from the physicist's instrumentation to the chemist's analytical tool. While

the pioneers of American mass spectrometry, Arthur Dempster, Walker Bleakney,

Kenneth Bainbridge, Alfred Nier, and John Tate researched elemental composition and

pure compounds, a need arose to analyze crude samples in the petroleum industry.6 For

this need to be met, new ionization techniques were required.

Electron Ionization

Initially, electron ionization (EI) became the standard for hydrocarbon analysis in

the petroleum industry, producing fragments for structural identification and molecular

pattern recognition. Electron ionization is widely used in organic mass spectrometry, and

is suitable for volatile and thermally stable molecules. El is a technique that uses









energetic electrons to "hit" molecules and remove an outer electron, forming a radical

ion. Devised by Dempster and improved by Bleakney and Neir, a diagram for a typical

El source is shown in Figure 1-1.7-8


filament
electron beam
-lenses for accelerating
/ and focusing ions
ion U= 70 V
I
volume e I

Sion beam
repeller -i .- ion beam
------ -to mass
Sanalyzer

Ion source
ooc electron trap
block



Figure 1-1. Diagram of an electron ionization source. Adapted from reference 9.

If an electron (e) transfers enough energy to a neutral molecule (M), exceeding its

ionization energy, ejection of an electron generates a radical cation (M+*):

M +ee M" + 2e- (1-1)

Electrons are emitted from an electrically heated filament and accelerated to 70 eV by

potential gradients. As sample molecules, typically vapor, enter the ionization region, the

electron beam collides with the molecules, resulting in deposition of energy. Most

organic molecules only require from 8 eV to 12 eV for ionization; therefore, the

additional energy retained in the molecule causes fragmentation. Figure 1-2 shows how

ion yields vary with electron energy. The decreased ionization efficiency at the lower

potentials is due to inefficient collisions; the ionization efficiency also declines at higher

potentials due to the collision efficiency. As the electron energy increases, the molecules

start to become "transparent", thereby lowering collision probabilities. Since each









electron has an associated wavelength (De Broglie wavelength), as the electron energy

increases, the wavelength decreases, diminishing the possibility for energy transfer.10






(D ) 10 C2H2
(D O) 1



"0 c u.


(10 50 102 102 10
10-
10 5 10 1031






Electron energy (eV)

Figure 1-2. Plot of ion yields versus electron energy. A maximum ion yield occurs near
70 eV. Adapted from reference 10.

Ionization techniques must consider both the internal energy transferred and the

physicochemical properties of the analyte. Some processes are energetic causing

excessive fragmentation; others produce mainly molecular ions. In electron ionization,

some classes of compounds have a critical energy for fragmentation that is extremely

low, such that no molecular ions are produced. Lowering electron potentials only

decreases overall ionization efficiency. It quickly became clear that a new methodology

would be needed for molecular ion formation.
V-C


10-2
10 50 102 103 104
Electron energy (eV)

Figure 1-2. Plot of ion yields versus electron energy. A maximum ion yield occurs near
70 eV. Adapted from reference 10.

Ionization techniques must consider both the internal energy transferred and the

physicochemical properties of the analyte. Some processes are energetic causing

excessive fragmentation; others produce mainly molecular ions. In electron ionization,

some classes of compounds have a critical energy for fragmentation that is extremely

low, such that no molecular ions are produced. Lowering electron potentials only

decreases overall ionization efficiency. It quickly became clear that a new methodology

would be needed for molecular ion formation.









Chemical Ionization

To understand electron ionization mass spectra, the factors affecting ion

fragmentation must be considered. As advances in ion decomposition theory led

chemists to recognize carbon-carbon cleavages and rearrangement mechanisms,

energetic became the common theme for explaining ion formation, ion fragmentation,

and gas-phase interactions.ll Ion formation relates to the energy required to produce an

ion from a neutral molecule. Ion fragmentation refers to the energy required to break a

chemical bond. Ion interactions involve the energies associated with collisions of ions

with neutrals, ions, or surfaces. The fundamental studies of ion-molecule interactions

enabled an understanding of gas-phase chemistry, allowing the development of

fragmentation limiting ionization techniques (e.g., chemical ionization).

Chemical ionization (CI) produces ions with little excess energy using collisions of

the analyte molecules with primary ions created in the source.12 Ion-molecule collisions

allow for a more controlled energy transfer process, reducing fragmentation and

producing intact molecular ions. The reduction of fragmentation with the production of

molecular ions is termed "soft" ionization. A typical CI source is shown in Figure 1-3.

The CI source, shown in Figure 1-3, uses an electron ionization filament to ionize

the reagent gas, which is leaked into an evacuated chamber. The reagent ions formed

interact with the neutral gas-phase analyte yielding positive and negative ions of the

sample. The ion-molecule reactions in the CI technique (i.e., proton transfer, charge

exchange, and others) depend on the properties of both the reagent and analyte.12

Consequently, the choice of both becomes important in the analysis process. Briefly,

some aspects of the analyte and reagent population are described below.










filament
electron beam
L / lenses for accelerating
= 2 and focusing of ions
U= 200V

I I
reagent gas I ion beam
-- ----------- >
i F to mass
repeller I analyzer

Ion source
electron trap
block



Figure 1-3. Diagram of a chemical ionization source. Adapted from reference 9.

A proton transfer reaction can occur in CI providing the proton affinity (PA) of the

analyte (A) is greater than that of the reagent ion (B):

BH +A -AH +B (1-2)

A caveat does exist. If the proton transfer reaction is highly exothermic, AH = PA(B) -

PA(A), the excess internal energy will promote fragmentation, preventing maximum

quasi-molecular ion formation [A+H]+. Reagent ions have characteristic proton affinities

listed in Table 1-1.12

An additional mode of ionization, in CI systems, is charge exchange. In this case,

if the recombination energy (RE) of the reagent ion (B) is greater than the ionization

energy (IE) of the analyte, an exothermic reaction proceeds:

B '+ A A +B (1-3)

The RE of the reactant ion is defined as the exothermicity of the gas phase reaction:

B+ + e-B (1-4)









For a charge exchange reaction to occur, proton transfer reactions cannot be

prominent. Proton affinities for larger organic molecules vary from approximately 160 to

240 kcal mol1.



Table 1-1. Common reagent gases for chemical ionization techniques.


Reagent Gas Reactant Ion (BIt)

CH4 CH5+

CH4 C2H5

H20 H+(H20)n

CH30H H+(CH30H)n

C3H6 C3H7

NH3 H+(NH3)n


Proton Affinity

131.6 kcal mol-1

162.6 kcal mol-1

166.5 kcal mol-1

181.9 kcal mol-1

179.5 kcal mol-1

204.0 kcal mol-1


*Stable reactant ions and their proton affinities are listed. Degree of solvation
depends on partial pressure of reagent gas. Thermochemical data for monosolvated
proton.12


Electron ionization provides the information for structural elucidation that is

necessary in the analysis of hydrocarbons and simple organic molecules. Chemical

ionization yielded complementary information with molecular ion production for high

proton affinity or low ionization energy molecules.

These two techniques, El and CI, allowed chemists to accept mass spectrometry as

a viable analytical technique; however, they do not provide an avenue to ionize more

fragile, larger molecules.









Biomolecule Analysis

As the biochemical and biological fields progressed, the need to measure molecular

weights of biopolymers became increasingly important. Instead of a more direct

measurement, molecular weight was determined by electrophoretic, chromatographic,

and ultracentrifugation methods based upon the molecule's conformation, Stoke's radius,

and hydrophobicity, respectively.10 El and CI require molecules to be in the gas-phase,

so they are only amenable to volatile thermally stable compounds. In some cases,

compounds suitable for derivatization can also meet those requirements.12 The study of

proteins, carbohydrates, cell membranes, and other large biological molecules

necessitated ionization techniques capable of producing molecular ions of fragile

thermolabile molecules. This need led to the eventual development of energy-sudden

methods.

Energy-Sudden Approach

Once again fundamental ion formation studies helped expand ionization techniques

into new territory. In this case, the understanding of energy-sudden techniques was

derived from the fundamentals of ion kinetics, with the basis lying in decomposition and

desorption kinetics.

Rapid heating. If desorption and subsequent ionization take place before

decomposition, a limited amount of fragmentation will occur.13 This competitive notion

for evaporation and decomposition yielded the idea of "rapid heating". The rate at which

energy is deposited into a sample affects the production of neutral gaseous molecules

over fragments. If a given compound AB is heated, it is assumed that AB will be

released in the gas phase and will fragment into A and B. The two processes,

vaporization and decomposition, can be written as shown in Equations 1-5 and 1-6.14









AB K AB (1-5)

AB KD AA+B (1-6)

The Arrhenius equation allows the rate constants of the reaction to be viewed as

logarithmic expressions as shown in Equations 1-7 through 1-9.

k =f exp(-E/RT) (1-7)

InK, = nF E/RT (1-8)

InKD = In F E/RT (1-9)

For thermally labile compounds that readily decompose, the rate constant for

decomposition (KD) is larger than for vaporization (K,) at low temperatures. Because the

activation energy for vaporization (E,) is higher than for decomposition (ED), the slope of

the vaporization reaction is steeper than that of the decomposition reaction. Figure 1-4 is

a plot of In k versus 1/Tfor decomposition and vaporization reactions.

If the relationships based upon decomposition and vaporization hold true, then at

high temperatures, where 1/Tis small, vaporization is favored over decomposition. In

other words, reaching the maximum temperature as quickly as possible provides a high

degree of desorption and limits fragmentation.



Plasma Desorption

Desorption techniques were the energy-sudden ionization methods developed to

produce molecular ions from compounds considered intractable (i.e., nonvolatile and

thermally unstable molecules). One of the first, demonstrated by MacFarlane and

Torgerson, was plasma desorption mass spectrometry (PDMS).15-16










Higher temperatures, Lower temperatures,
vaporization favored decomposition favored


In kDecomposition reaction


Vaporization

Ev> ED

1I/T

Figure 1-4. Plot of rate constants for decomposition and vaporization versus 1/T.
Adapted from reference 13.



Desorption and ionization occurred using energetic fission fragments from a radioactive

californium source (252Cf). Califomium-252 results in primarily (97%) emission of alpha

particles, yet it also undergoes (3%) spontaneous fission, emitting two multiply charged

fission fragments simultaneously and in nearly opposite directions. Typical decays

involved 106Tc and 142Ba with energies of 104 and 79 MeV. The ionization technique

works by using the pair of fission fragments to provide high energy collisions. One

fragment penetrates a thin metal foil, releasing a burst of secondary electrons that begin

the instrumentation timing sequence. The second fission fragment penetrates an

aluminum foil holding the sample. Ions desorbed from the sample are accelerated to

energies of 10 to 20 eV, pass through a drift tube, and are detected. Alpha particle

emission is discriminated against by the production of lower kinetic energy (-4 MeV)

secondary electrons. Commercialized by Bio-Ion Nordic (Uppsala, Sweden), PDMS










could analyze small proteins up to -20 kDa.17 A diagram of the source is shown in

Figure 1-5.



fission fragments
accelerated ions



TOF drift region
fission 252Cf support Ion
fragment fission foil with acceleration Analyte
detector source sample grid detector
layer



Figure 1-5. Diagram of californium plasma desorption ionization source. Adapted from
reference 18.



Fast Atom Bombardment

Next in the expansion of energy-sudden ionization techniques was fast atom

bombardment (FAB). Developed by Barber, FAB uses high energy (5 keV) neutral

atoms to impart energy onto a target, where a non-volatile liquid matrix (e.g., glycerol,

m-nitrobenzyl alcohol) contains analyte.19 As the energetic atoms, typically argon, hit the

sample surface a shockwave is induced that ejects ions and molecules.20

The development of FAB was partially to circumvent problems with electrostatic

charging upon ion impact in secondary ion mass spectrometry (SIMS), which disturbed

ion source potentials.2122 SIMS uses a focused ion beam to cause secondary ions to be

emitted from a sample surface. Using the FAB technique, intact molecular or

quasimolecular ions could be generated even in the case of highly polar compounds,

which are known to be poor candidates for electron and chemical ionization.

Additionally, the use of a liquid matrix in FAB decreased the rapid decomposition









characteristic of the harsh conditions in desorption/ionization for certain samples.2324

Using a FAB ion source, molecules nearing 10 kDa can be observed. A diagram of a

FAB ionization source is shown in Figure 1-6.

Ion formation theories for FAB consist of the chemical ionization model and the

precursor model.25-27 The chemical ionization model assumes formation of the analyte

ions in the region directly above the liquid matrix, referred to as the selvedge region. In

this space, a plasma state similar to the reagent gas plasma in chemical ionization can

exist due to the ions created from the supply of impacting primary atoms. Constituents in

this region would undergo numerous reactions, including the protonation of analytes to

yield quasimolecular ions (i.e., [A+H] ). While matrix molecules are preferentially

ionized for statistical reasons, they may act as the reagent ions in a system mimicking

chemical ionization.

The precursor model of FAB mostly applies to ionic analytes or samples that are

easily converted to ions in the liquid matrix. The model suggests that ions are preformed

in the matrix and are merely transferred to the gas-phase. Support comes from

observations that decreasing pH increases protonated analyte ion yields. Additionally,

relative intensities for protonated ions seem independent of partial pressure of amines in

the gas phase, and dependent on acidity of the matrix.28 Chemical ionization reactions

would suggest just the opposite. Furthermore, incomplete desolvation of preformed ions

would explain observed matrix adducts [A+Matrix+H].

Although the liquid matrix provides a fresh surface layer for ion production by

convection and diffusion, it does have additional requirements.22'29 The matrix must: (1)

absorb the primary energy, (2) solvate the analyte, (3) have a low vapor pressure, and (4)










assist in analyte formation by yielding proton donating/accepting species upon

bombardment. While a matrix can be adapted as analytes require, the wrong matrix may

result in complete signal suppression.22


electron ionization
of FAB gas



acceleration and
focusing of
primary ion beam




neutralization of
energetic ions


beam of .I -


- anode

filament


FAB gun





Xe gas supply
al gas



secondary ions to
mass analyzer


ion volume lenses for ion
acceleration


Figure 1-6. Diagram of a fast atom bombardment ionization source. Adapted from
reference 9.




Laser Desorption Ionization

Before the advent of plasma desorption and fast atom bombardment, laser

desorption/ionization (LDI) sources were used to analyze low-mass organic salts and

light-absorbing organic molecules.30 While cases did exist for LDI to obtain mass spectra









of biomolecules, the analysis of fragile compounds over 1000 Daltons was not

routine.9'31-32 This allowed FAB and 252Cf-PDMS to be far more effective in generating

bimolecular mass spectra.

A number of laser systems were used for desorption techniques, yet infrared (IR)

lasers (e.g., CO2 and Nd:YAG) saw extended use and provided mechanistic explanations

of the process.33 With instantaneous pulse energies of 100 mJ, and focused beams giving

fluence values of 1 MW.cm-2, thermal mechanisms were predicted. As evidence, neutral

and alkali species were observed to be desorbed from the sample beyond the actual laser

pulse length.31 This indicated that as the sample was heated, thermal desorption allowed

ion emission. Additionally, at longer delay times, IR LD produced lower kinetic energy

ions with less fragmentation.31 Since there was also no apparent wavelength dependence,

a thermal process fit the observations.33

Although time widths of the laser pulse ranged from nanoseconds to microseconds,

and rapid heating seemed possible, the problem with laser desorption was that higher

temperatures could not be reached quickly enough to obtain intact molecular ions. What

was finally needed for the method to succeed was a medium that enabled the conversion

of the irradiated photons to thermal energy without directly heating the analyte.

Matrix-Assisted Laser Desorption/Ionization

A major change in mass spectrometry occurred with the addition of light-absorbing

compounds to sample mixtures, allowing a controlled desorption/ionization event. Two

matrix mixtures that allowed photon absorption were originally developed: (1) ultra-fine

cobalt particles, glycerol, and analyte; and (2) a co-crystallization of analyte with organic

matrix.13,34-37 While both methods are capable of producing mass spectra of large fragile

proteins (> 100 kDa), the use of the cobalt particles is considered the first ionization









method that allowed the mass spectrometry community to think about analyzing

thermally labile biomolecules. This is why Koichi Tanaka was awarded a portion of the

Nobel Prize in 2002.13 Using the absorbing particles allowed the collection of some of

the first large biomolecule mass spectra.38 However, the co-crystallization technique

developed by Hillenkamp and Karas using light-absorbing organic matrices has been the

more prolific method for biomolecule analysis.39

The co-crystallization method, referred to as matrix-assisted laser

desorption/ionization (MALDI), uses a variety of light-absorbing matrix molecules to

control uptake of laser irradiation and cause desorption and ionization of the analyte.40

Unlike the wavelength independence seen in LDI, MALDI was developed from the

wavelength dependence of tryptophan analysis.35 In the simple system, the amino acid

acted as the absorbing molecule. Hillenkamp later reported the matrix-assisted technique

of molecular ion desorption with the use of a more traditional nicotinic acid matrix.41

The components of a MALDI source are straightforward. Figure 1-7 shows the general

configuration for the ion source.

The source comprises a laser, sample plate (with sample), and acceleration field for

transfer into the mass analyzer. While the source is simple in construction, the

underlying processes for desorption and ionization are less than trivial.42-46 The critical

parameters involve a minimum of laser wavelength, laser fluence, matrix formulation,

and sample preparation. Additionally, the mass analyzer, ion transmission, and analyte

parameters must be considered. The many variables to consider during the MALDI

process have made mechanistic theories difficult to produce; therefore, the overall

mechanism for ion formation is still a subject of continuing research.









An understanding of ionization pathways could help to maximize ion yields,

control analyte charge states and fragmentation, and gain access to new classes of

analytes. Knowledge of the ion formation process may also provide rational guidelines

for matrix selection, something treated more as an empirical art than a scientific process.





Splasma
0o E *
S( D- :D. to mass
/. analyzer
sample *I 3 **
acceleration


les laser pulse
ens \



Figure 1-7 Diagram of a matrix-assisted laser desorption/ionization source. Adapted
from reference 41.

Mechanisms

Presently, no single chemical or physical pathway explains all positive and

negative ions in the MALDI spectrum.43 Since experimental variables drastically affect

the mass spectra outcome, several mechanistic theories have been produced to describe

each effect. The mechanisms can be divided into two categories: primary and secondary

ionization.43'45-46 Primary ionization refers to the generation of the first ions from neutral

molecules-often matrix-derived species. Secondary mechanisms involve the ions not

directly generated by primary processes-usually analyte ions.









Major primary mechanisms include: single molecule multi-photon ionization,

energy pooling, excited-state proton transfer, disproportionation reactions, desorption of

preformed ions, thermal ionization, and spallation. Major secondary ionization

mechanisms include: gas-phase proton transfer and gas-phase cationization.

Primary Ionization Reactions. Since the wavelength dependence of MALDI

assisted in its eventual discovery, laser wavelength plays an important role in many

mechanistic theories. With the matrix requiring energy absorption, the laser wavelength

must be matched with the matrix chromophore, the most common is 337 nm from a

nitrogen laser; however, Nd:YAG harmonics, eximer lines, and infrared lasers have been

employed. Table 1-2 shows typical photon energies and wavelengths for MALDI laser

systems.43

Table 1-2. Typical laser wavelengths, photon energies, and pulse widths used for
MALDI.43


Laser

Nitrogen

Nd:YAG x3

Nd:YAG x4

Excimer (XeC1)

Excimer (KrF)

Excimer (ArF)

Er:YAG

CO2


Wavelength

337 nm

355 nm

266 nm

302 nm

248 nm

193 nm

2940 nm

10600 nm


Photon Energy (eV)

3.68

3.49

4.66

4.02

5.00

6.42

0.42

0.12


Pulse Width

4 ns

5 ns

5 ns

25 ns

25 ns

15 ns

85 ns

-1 Ls









Even with the importance of the wavelength/matrix combination, MALDI spectra

do not show significantly different ions with different laser wavelengths.47 However,

alterations in pulse energy and sample composition are necessary to obtain similar ion

populations.48

Multi-photon Ionization. Multi-photon ionization explains the radical cations

formed through the wavelength dependence of MALDI.35'49-50 The reaction shown in

equation 1-10 produces matrix (M) radicals, which could be key intermediates for analyte

ions.

Sn(h) M +e (1-10)

The criticism for the mechanism lies in the energetic needed to ionize the matrix

molecules. Two photons from a nitrogen laser yield 7.36 eV; however, the ionization

potentials (IP) for common matrix molecules are higher (i.e., DHB at 8.05 eV).43 The

typical irradiances values, 106-107 W-cm-2, make three photon ionization unlikely.51

Recent experiments suggest that clustered matrix molecules may have lower IP, yet

questions still remain about the accuracy of the solid matrix measurements.43

Energy Pooling. Direct multi-photon ionization may not seem plausible due to

energetic, but the excited states of matrix molecules are considered a viable starting

point.52-53 A possibility is that two or more separately excited matrix molecules "pool"

their energy to yield one matrix radical cation.54-55 The reaction pathways for this

"energy pooling" mechanism are shown in Equations 1-11 and 1-12.

AMM M*M*-M+M+*+e (1-11)

M*M +A MM + A+' + e (1-12)









Equations 1-11 and 1-12 could explain the fluence dependence associated with

MALDI. A critical factor in the desorption/ionization process is the energy density

supplied to the sample (fluence, J-cm-2); however, alterations in the irradiance (W-cm-2)

seems to have less of an effect on the mass spectra.56-58 Fluence versus irradiance

dependence indicates that the number of photons delivered is important, not the rate at

which they arrive.

Excited-state proton transfer. Furthering the excited-state theories is the excited-

state proton transfer mechanism, which helps explain protonated species.36 The pathway

is shown in Equations 1-13 to 1-15.

M + hv M (1-13)

M*+A ->(M -H) +AH+ (1-14)

M*+M->(AM-H) +MH+ (1-15)

Most matrices are not known to be good excited-state proton transfer agents.4951

However, without a better knowledge regarding the local environment in a MALDI

sample, refuting the mechanism is difficult.

Disproportionation. Each laser pulse in MALDI yields both positive and negative

ions. To explain this observation, disproportionation reactions have been suggested.59

The pathway is shown in equation 1-16.

MM 2h (MI)* ->(M -H) +MH+ (1-16)

The problem with the mechanism is that positive and negative ions are not

correlated in mass spectra. The fluence thresholds for each ion polarity are also different,

suggesting alternate pathways.









Preformed ions. Matrix solutions are typically made from organic acids;

therefore, it is realistic to expect that preformed ions may exist in the sample. For ionic

compounds, the idea that preformed ions are desorbed is reasonable, yet it is difficult to

be certain whether the ions observed are truly preformed or the result of secondary gas-

phase reactions discussed in the following section.60-62

Physical mechanisms. Chemical ionization mechanisms have dominated the

MALDI literature, but physical mechanisms have also been considered. Both thermal

ionization and spallation (structural fracture of the solid) have been suggested for IR

MALDI.57,63 Using infrared lasers, penetration depth is much greater, which may result

in mechanical stress.64 Additionally, since IR absorptions are weaker, the energy per

volume deposited is typically too low to fully "melt" the material desorbed. Hillenkamp

has proposed that spallation is an important mechanism in this case.63 Thermally induced

stress that builds faster than can be dissipated leads to a mechanical failure of the solid

and ablation of material without direct vaporization.64

Secondary ionization. Molecular dynamics simulations have proposed that the

MALDI plume after laser desorption is a dense cloud containing single molecules, ions,

and clusters.66-67 Thus, an impenetrable plume of material provides the opportunity for

primary ions to undergo ion-molecule reactions, necessitating secondary ionization

reactions.68

Proton transfer. If primary ions are radical cations then proton transfer matrix-

matrix reactions can readily produce protonated matrix ions:49

M++M ->MH+ +(M-H)' (1-17)









This is similar to the proposed mechanism for protonated glycerol in FAB.69

Likewise, matrix-analyte reactions can produce protonated analyte ions, important in

MALDI spectra. Just as in chemical ionization mechanisms, the presence of protonated

analyte requires a proton transfer reaction.

MH+ A -M+ AH+ (1-18)

Again, as in CI the reaction proceeds when AG<0.12 Since proton affinities of

peptides and proteins are on the order of 240 kcal-mol-1, and most measurements of

MALDI matrix proton affinities are between 183-225 kcal-mol-1, the reaction should be

thermodynamically favorable.7071 It has also been noted that protonated analyte ion

intensity increases for basic residue peptides indicating a chemical ionization approach to

protonation.62 Additionally, varying matrices with a standard analyte alters the internal

excitation available (due to proton affinity differences) and affects the degree of

fragmentation. This has been referred to as "hot" and "cold" matrices.772'76

Cationization. Gas-phase cationization as a secondary ionization mechanism

describes abundant cationized adducts in MALDI spectra.73-74 Ion-molecule reactions of

this type have also been proposed to explain the pseudo-molecular ion formed by laser

desorption mass spectrometry without a matrix.63 Studies have shown that salts added to

MALDI samples allow the cationization of synthetic polymers, adding evidence to the

mechanism.75 Similar to protonation reactions, cationization requires cation affinities of

the analyte to exceed that of the matrix.

Matrix considerations

Even with numerous mechanistic studies, matrix choice is not systematic.

Difficulties in analysis stem from both ionization and co-crystallization issues.43 The









appendix contains a list of some common MALDI matrices and their corresponding

analyte classes. Most use pulsed UV lasers, and consequently are UV absorbers. The

matrices are derivatives of benzoic acid, cinnamic acid, and related aromatic

compounds.76

Even when the appropriate laser/matrix system is chosen, matrices need further

development for specific analyte applications. In MALDI sample preparation, peptides

and proteins are generally solubilized in 0.1% aqueous trifluoroacetic acid at a

concentration of approximately 10-5 M.42 One microliter of solution is then mixed with a

saturated aqueous matrix solution (around 10-3 M), which is allowed to evaporate

forming crystals. Additional crystallization techniques are described in the appendix.



Time-of-Flight Mass Spectrometry

Historical Perspective

With the development of plasma desorption, fast atom bombardment, laser

desorption ionization, and matrix-assisted laser desorption/ionization, the challenge in

mass spectrometry became not the production of ions but rather development of the mass

analyzer. The first mass spectrographs devised by Thompson, Aston, and Dempster

utilized magnetic and electrostatic fields for ion separation.2 While these configurations

are still used as today's high resolution double-focusing spectrometers, research focusing

on the reduction of magnetic fields provided the most widely used MALDI coupled mass

analyzer. As magnets became a limiting factor in mass analyzer construction, primarily

due to size and cost, W. E. Stephens devised an analyzer that did not require magnetic

fields. In 1946, Stephens stated:77










"Such a mass spectrometer should be well suited for composition control, rapid
analysis, and portable use. Magnets and stabilization equipment would be
eliminated."

He described the mass analyzer, the time-of-flight (TOF) mass analyzer, in his patent

78
as:

"...apparatus for obtaining pulses of ions segregated according to mass-velocity
relationships, collecting the ions to obtain pulses of current dispersed in time and
recording the result. Separation of ions of different masses does not depend upon
slit width as in the case for a conventional mass spectrometer, but depends upon
only the path length, the accelerating voltage, pulse length and the detecting
device."

Time-of-flight Theory

The basic principle of the time-of-flight mass spectrometer (TOFMS) is the

measurement of time as an ion travels a fixed distance. The time is related to the ion's

mass-to-charge ratio. The simplest time-of-flight consists of a source extraction region, a

drift region, and a detector. A diagram for a TOF is given in Figure 1-8.

In the source region, a voltage is applied to a backing plate that accelerates ions to a

final kinetic energy (eV):31

1 (1-19)
KE = my
2

mv2 (1-20)
= eV
2

The mass (m) and velocity (v) of the ion are then related to the energy it obtains.

Dimensional analysis can be conducted by considering units of kg for mass, m-s1 for

velocity, and using 1.60 x 10-19 J-eV-1. The drift region is field free, so the ions cross the

region with constant velocities that are inversely proportional to the square root of their

31
masses.













Drift Space


L (Drift Length)


Grid


!9
9,


Detector 0 TOF




0 TOF


Sample
Plate



rv


*




*


Drift Space
4


Figure 1-8. Diagram demonstrating the principle theory in time-of-flight mass analysis.
Adapted from reference 31.



2eV 1/2 (1-21)
v=--

Thus, light ions travel faster and arrive at the detector sooner. Ion flight times fall in the

range of 10 to 200 [is depending on the spectrometer arrangement. Flight time (t) is then

related to velocity by the length of the drift tube (D).31 Typical units are s for time and m

for tube length.


Sample


M + M1 peak

0 TOF









S1/2 (1-22)
2eV

A general derivation of the ion flight time should include the time the ions spend in

the source region, yet if the region is short with respect to the drift tube the resulting

equation is essentially the same as equation 1-22.

Relating flight time to molecule mass requires a mass scale that follows a square-

root law. The linear equation:

t= am/2 +b (1-23)

allows masses to be determined with as little as two known masses. While constant a

relates to flight tube length and acceleration voltages, constant b takes into account time

offsets due to the laser or detector.

The mass resolving power for a mass spectrum is defined as m/Am.79 In a time-

of-flight analyzer this equates to a temporal resolving power as follows:

m t (1-24)
Am 2At

where At is commonly measured as the full width at half maximum (FWHM). The basic

resolution equation is derived from rearrangement of equation 1-22:

(2eV 2 (1-25)




Kinetic energy spreads

Equation 1-25 is derived from TOF theory; however, deviations in kinetic energy

and spatial formation exist.80 Ions are generally formed with some initial kinetic energy,

so KE = eV + Uo, where Uo corresponds to the initial kinetic energy. Kinetic energy









spreads can account for velocity differences in the ions, detrimental to spectral resolving

power.83 Resolving power enhancements can be seen by increasing accelerating voltages

(i.e., 3-30 kV), giving the ions a total energy much higher than the initial energy. Time-

lag focusing is also used to reduce the initial kinetic energy spreads.85 Developed by

Wiley and McLaren, time-lag focusing provides a time delay between the ionization

period and the ion-extraction pulse. This enables ions to drift within the field-free source

before extraction, allowing them to distribute according to their initial kinetic energy.31'80

The distributions are converted to spatial spreads. One drawback for time-lag focusing is

its mass-dependent nature.31 Only a narrow range can be focused for a particular value of

time-delay.80'85 Further minimization of the energy distributions, sometimes inherent in

the ionization technique, can be done using a reflectron.31'84'88 The simplest of reflectrons

acts as an electrostatic ion mirror, enabling the ion kinetic energy to be converted into

penetration depths. Longer times in the reflectron yield the same total time-of-flight for

isobaric ions due to their increased kinetic energy and shorten drift region times.

Spatial spreads

Spatial distributions occur when ions are formed in different regions of the source,

and then are accelerated through varying distances in the extraction field, resulting in

higher drift velocities.31'85 Using a uniform accelerating field in the extraction region

yields a plane, the spatial focus plane, located a distance twice that of the extraction

region. This is where isobaric ions of differing velocities would be focused.85 In this

arrangement, the spatial focus plane is typically not located at the detector, which would

minimize spatial spreads. To move the focus plane closer to the detector, a two-stage

extraction region is used.31'80'81 Additional flight time spreads can also be caused by









turn-around time. This is the time ions having initial velocities in the opposite direction

of the flight path spend in the source.

Since some time spreads cannot be prevented, and because time distributions (At)

are maintained as the ions approach the detector, mass resolving power is limited.

However, considering the resolving power equation for time-of-flight mass

spectrometers, t/2At, an increase in total time, can increase resolving power with a

constant time spread. This equates to using longer flight tubes for further

enhancements.86

Desorption Ionization Techniques

Using a gas-phase ionization technique, such as El or CI, with a linear TOF-MS

requires either pulsing of the ion source or accumulation of the continuously formed ions

before the extraction pulse.31'8 Yet, with a desorption/ionization technique (i.e.,

MALDI) the system is simplified. Since the sample is placed on a surface, spatial

distributions and ion turn-around time are less significant. The ions are formed on a plate

parallel to the detector. Additionally, with plasma desorption occurring within 10-9 s of

impact, and MALDI generally using laser pulse widths of 3 to 100 ns, ionization times

are shorter than the drawout pulse resulting in minimal initial temporal distributions.31

The first commercial time-of-flight mass spectrometer was produced by the Bendix

Corporation (Detroit, MI).2 The system had a mass range (at repetition rate of 10 kHz) of

about 400 amu, and a mass resolving power of 200. The Bendix spectrometer was the

platform that allowed the addition of multiple ionization techniques to be examined with

TOF. At this early stage, success of the TOF was limited by both its mass range and

mass resolving power. Combining the use of pulsed ionization techniques with boxcar

recording methods together produced an extraordinary low duty cycle. It was not until









the further development of detectors, data acquisition systems, and the coupling of

desorption ionization techniques did TOF mass analyzers become standard equipment.82

Today, time-of-flight mass spectrometers can obtain resolving powers of over 10,000

with detection limits near attomole range.42'87



Conclusions

Each developmental stage of an ionization technique or mass analyzer affords new

analytical opportunities. While the recent advances in MALDI and TOF allow for the

routine study of biomolecules, subtle alterations (atmospheric pressure and orthogonal

geometry) also present additional advantages. The research in this document involves the

fundamental and practical study of the processes in an atmospheric pressure MALDI

source coupled to a TOF mass spectrometer. The focus is on liquid matrices, their

interactions at atmospheric pressure, and the prospective analytical utility they provide.














CHAPTER 2
DESIGN OF AN ATMOSPHERIC PRESSURE MATRIX-ASSISTED LASER
DESORPTION/IONIZATION SOURCE FOR AN ORTHOGONAL-ACCELERATION
TIME-OF-FLIGHT MASS SPECTROMETER

Introduction

"As a pulsed technique, MALDI is easily compatible with time-of-flight mass
spectrometry and has been responsible (more than any other technique) for the
renewed interest and active development of this mass analyzer."
-Robert Cotter31


A passage from Robert Cotter's 1997 book Time-of-Flight Mass Spectrometry

reflects the general scientific community agreement on matrix-assisted laser

desorption/ionization (MALDI) and mass spectrometry instrumentation. Therefore, it's

ironic that recent developments in time-of-flight (TOF) instrumentation have been

directed towards the use of continuous ionization sources.



Background

Orthogonal-Acceleration Time-of-Flight

Orthogonal-acceleration time-of-flight mass spectrometry (oa-TOFMS) uses ion

beam deflection techniques.88 In an oa-TOFMS, mass analysis is done orthogonall" to

the ion source axis. Older instruments have used beam deflection techniques to narrow

the ion packets pulsed into the flight tube. With orthogonal geometry, developed by

O'Halloran, deflection is taken to the extreme (900), yielding an ability to sample

continuous ion sources.88-90 For years there was minimal interest in orthogonal geometry

instruments, until the configuration's rediscovery by Dawson, Guilhaus, and Dodonov.91-









92 Today the configuration provides numerous benefits to the TOF instrumentation

community.

Orthogonal-acceleration TOFs offer improved mass resolving power, duty cycle,

and compatibility with continuous high-energy spread ions (e.g., electrospray ionization

(ESI)). With orthogonal deflection a new component of velocity, which is independent

of initial ion source velocity, is added to the sampled ions.88 The decoupling of mass

analysis from the ion source leads to reduced time distributions for the ions. Instead of

the initial kinetic energy distributions (due to ionization processes) affecting mass

resolving power, orthogonal sampling discriminates against energy spreads in the ion

source axis. Axial spreads are then distributed perpendicular to the pulse-out electrode of

the flight tube, and do not affect time resolution. Provided the detector is large enough,

the ion spread in the axial direction will disperse over the detector plate of the oa-TOF.

While the oa-TOF provides a duty cycle advantage, the term has been

misunderstood and at times referred to as "increased sensitivity." Duty cycle in mass

spectrometry is the ratio of the time ions are extracted for mass analysis over the total

time ions are produced. In a properly arranged oa-TOF, as an ion packet is mass

analyzed, a new packet is filling the pulser region of the flight tube.88 In theory this

allows for a 100% duty cycle, yet it says nothing about the analyzer transmission and

detector efficiency, the parameters needed to determine instrument sensitivity.

With the ability to effectively sample continuous ion sources, electrospray

ionization (ESI) was one of the first techniques adopted for the oa-TOFMS.93-94 ESI,

originally developed by John Fenn, is a process by which ions in solution are transferred

to the gas-phase with limited ion fragmentation.95-96 The coupling of ESI to oa-TOFMS









affords three main advantages: increased duty cycle, improved mass accuracy, and higher

m/z capability.88'97-98 While oa-TOF mass spectrometers were used for continuous

ionization source mass analyzers, MALDI sources were coupled to oa-TOFs for different

reasons. Unlike continuous sources, MALDI was compatible with the pulsed scheme of

linear TOF analyzers. However, MALDI generates large initial kinetic energy spreads

for ions, effectively reducing mass resolving power. While ion mirrors, reflectrons, and

delayed extraction correct for some energy spread distributions, improvements were

needed.99 Delayed extraction, for instance, must be tuned for each desired m/z range.31

Orthogonal-acceleration TOF spectrometers decouple the energy of the

desorption/ionization processes from mass analysis, thereby increasing mass resolving

power and mass accuracy.

Atmospheric Pressure Inlets

Coupling atmospheric pressure ionization (API) sources to a mass analyzer requires

a 107 to 108 reduction in pressure, demanding precise inlet designs and efficient vacuum

pumps. Since the sampling efficiency of an API source is dependent upon the number of

ions and the amount of gas that can be introduced into the orifice, multi-stage pumping

systems are routinely required. The gas expansion that occurs from atmospheric pressure

to vacuum, termed a supersonic jet expansion, also complicates the design of an API

source, creating two major consequences: (1) ions must be sampled with a skimmer cone,

and (2) ion-solvent clusters must be prevented.33

Skimmer cones are required due to the nature of free jet expansions. The

expansion of a gas from a high-pressure region into a low-pressure region through a small

nozzle produces a supersonic jet of gas with a narrow velocity distribution and a high

flux per unit area.100 The sonic jet travels at the local speed of sound in the gas.101 The









expansion generates a shock wave terminating at the Mach disk where gas slows and

diffuses. Extracting the sample through an orifice (skimmer) placed within the region

before the Mach disk, termed the zone of silence, allows for maximum ion collection.101

When a mixture of gas and ions is transferred from atmospheric pressure to low

pressure within the MS instrument, adiabatic cooling occurs as the mixture rapidly

expands in the vacuum.102 If polar neutrals (e.g., water or solvent) are present in the

mixture at that time, condensation of these neutrals on the analyte ions occurs. The size

of the formed clusters may then exceed the mass range of the analyzer and also lower the

analyte signal by distributing it over several ion signals. In modem MS instruments

designed for atmospheric pressure ionization (API), the problem with clustering is of

high concern. The most common methods to prevent large ion-solvent clusters from

entering the mass analyzer are the addition of: (1) an axial potential gradient, (2) a heated

bath gas, or (3) a counter-current dry bath gas. A potential gradient between the nozzle

and skimmer results in a field to accelerate the ions relative to the neutral carrier gas

molecules, producing energetic collisions that fragment the clusters.101-102 A heated bath

gas allows the temperature to remain above the condensation value during free jet

expansion. Finally, a counter-current dry bath gas provides cluster prevention by

removing solvent molecules, averting resolvation. A counter-current flow also limits

non-ionized material from entering the system, making it more tolerant to "dirty"

samples. While electrostatic and electromagnetic forces play a pivotal role in ion

transmission, high-pressure sources also introduce aerodynamic forces that affect ion

trajectories.









Atmospheric Pressure MALDI

The advent of the oa-TOF using an AP inlet allowed MALDI sources to be

operated at atmospheric pressure. Laiko and Burlingame were the first to examine

APMALDI sources for analytical purposes.103-104 Although the oa-TOF format provided

a logical means for examining the APMALDI source, additional groups coupled the new
105-107
source to commercial ion trap mass spectrometers.10107

APMALDI sources offer reduced constraints on ion source pressure. This can be

useful for high-throughput screening, where automation must be used for source

construction. Atmospheric pressure conditions also provide an opportunity to examine

volatile matrices or other vacuum incompatible samples. Furthermore, an APMALDI

source could be interchangeable with other AP sources on an atmospheric pressure

interface mass spectrometer. The last benefit, interchangeability, allowed the original

APMALDI investigators to form a company, Mass Technologies, directed at producing

commercial APMALDI sources for a variety of currently employed spectrometers.10s

With interchangeable ion sources a user can add utility to an already high cost

instrument. Each ionization process allows for a range of chemical classes to be ionized,

and in specific cases may provide complementary information. Figure 2-1 shows the

complementary nature of atmospheric pressure ionization sources for mass analysis. ESI

may dominate the liquid chromatography market due to eased liquid separations

coupling, but MALDI remains the method of choice for peptide/protein identification.

By most accounts, APMALDI spectra are "similar" to vacuum MALDI spectra.105

Therefore, our initial experimental goals were not focused on spectra evaluation, but the

opportunities available for additional analytical utility using APMALDI. Furthermore,








with the limited history of APMALDI, the study of desorption and ionization phenomena

at atmospheric pressure may assist in understanding MALDI.


Figure 2-1. Diagram depicting the complementary nature of atmospheric pressure
ionization sources is shown. Each technique, atmospheric pressure photo
ionization (APPI), atmospheric pressure chemical ionization (APCI),
electrospray ionization (ESI), and matrix-assisted laser desorption/ionization
(MALDI), has a selective region of polarity and molecular weight in which it
is most effective. Adapted from reference 109.

Fundamental research in MALDI has become more of a topic for scientific

discussion, as one of the founders of MALDI, Franz Hillenkamp, recently wrote, "...it is

time to go back and do some more basic research."110 The beginning of these studies

required the design, construction, and optimization of an APMALDI source. This

chapter describes the efforts to couple an APMALDI source to an oa-TOFMS.


-200,000



- -15,000






o 0
n APCI




Polarity










Experimental Methods

Mass Spectrometer

The mass spectrometer used is an orthogonal-acceleration time-of-flight (oa-TOF).

Figure 2-2 is a diagram of the oa-TOF.


Signal Conversion Electronics

Multiple Anodes


Turbomolecular ions _
Pumps
SFlight
Tube
Skimmer Heaviest
Sr r ons
Nozzle
RF
Quadrupole



LPulsing region

Einzel Lens



Figure 2-2. Diagram of the orthogonal-acceleration time-of-flight mass spectrometer used
in the studies presented is shown.

The spectrometer is a prototype developed by LECO Corporation (St. Joseph, MI,

USA). Originally designed for an ESI source, the spectrometer uses a heated curtain gas

for ion cluster prevention. Once the ions enter the 254 [m nozzle orifice, they are

sampled through a skimmer cone (2 mm inner diameter) located -6 mm away. Voltages

for the nozzle and the skimmer cone can be altered independently; the pressure in the

region between the two components is -3 Torr. This region is pumped by a two stage









rotary vane roughing pump (E2M18, 4.7 L-s-1, BOC Edwards, Willmington, MA, USA),

which also backs two turbomolecular pumps. Next, the ions travel into an RF-only

quadrupole. The RF supply runs at a fixed 1 MHz frequency with the quadrupole also

having an adjustable DC offset of 0-300 V. A DC gradient of 0-50 V can also be applied.

The region within the quadrupole is maintained at a pressure of -0.1 Torr by the second

stage of a hybrid turbomolecular drag pump (TMH261, 210 L-s-1, Pfeiffer, Germany).

The primary stage of the hybrid pump is connected to the flight tube. Ions exit the

quadrupole region through a 2 mm by 0.5 mm rectangular slit. An Einzel lens focuses

and directs the ions into the flight tube pulser. A second turbomolecular pump is used to

evacuate the Einzel region (TMH71, 70 L-s-1, Pfeiffer, Germany). The pulser then

accelerates the ions up the flight tube (maintained at a pressure of 1.5 x 10-7 Torr). The

flight tube is 50 cm long and constructed of printed circuit board.1ll The tube is

segmented into 39 electrodes, from pulser to detector. Potentials are applied to the

electrodes through a voltage divider to form a parabolic potential field for the ions,

designed to improve mass resolution.

The detector used in the spectrometer is a chevron configuration dual microchannel

plate (MCP) assembly. Electrons from the MCPs (4 cm by 8 cm, Hamamatsu, Japan)

strike an array of 36 anodes. The detection system, termed a time-to-digital converter

multi-anode detector (TDC-MAD), provides rapid temporal resolution (1 ns) and

digitization rate (1 GHz) while allowing a large dynamic range from the multiple

anodes.112 The signals from the anodes are fed into comparators and then a combiner

board. The data from the combiner board is sent to a host board where successive spectra

are summed and sent to an array board for compression and transfer to a PC via SCSI









(Small Computer System Interface). The pulser is operated at a frequency of 5 kHz,

while the array board can output summed spectra at a rate varying from 0.1 to 100

spectra-s-1. This allows the summation of 50 to 50,000 pulses for each output spectrum.

Software, ChromoTofversion 3.21 Beta (LECO Corp., St. Joseph, MI, USA), allows

spectral viewing. The mass spectrometer provides both positive and negative ion

detection modes at a rated mass range of-1 to 6000 m/z. Resolving power is listed as

-2000 at m/z 600.

Electrospray Configuration

The ESI setup consisted of an uncoated pulled fused silica fiber with 30 [m inner

diameter and 360 [m outer diameter (FS360-100-30-N, New Objective, Woburn, MA,

USA). Figure 2-3 is an optical image of the ESI tip taken using a Charge-Coupled

Device (CCD) camera (5M, Pixera, Los Gatos, CA, USA) mounted on a microscope

(Edmund Scientific, Barrington, NJ, USA).

The flow rate, 0.5 .iL-min-1, was applied using a syringe pump (Pump 11, Harvard

Apparatus, Holliston, MA, USA). A voltage of 3250 V was applied to a liquid junction

contact by a power supply internal to the mass spectrometer. The needle was placed -10

mm from the orifice of the spectrometer and a curtain gas cover directed heated nitrogen

gas (80 C at 800 mL-min-1) towards the ESI tip. The voltage on the curtain gas cover

was set to 1550 V. Spectra were acquired at a rate of 4.17 spectra-s-1 (1200 pulsed

packets). Reserpine and verapamil solutions were 2 ng-iL-1 in 50% aqueous methanol

solutions with 1% acetic acid. All chemicals and solvents were purchased from Sigma

Aldrich (St. Louis, MO, USA) and used without further purification.

































Figure 2-3. A photograph of the electrospray tip used for mass spectrometer
characterization.

Table 2-1 lists the potentials of the ion transfer optics and the mass analyzer when

using an electrospray ionization source.



Table 2-1. Mass spectrometer conditions used to acquire electrospray spectra.


Nozzle 150 V
Skimmer 65 V
Quadrupole RF 300 V
Quadrupole High 41 V
Quadrupole Low 44 V
Quadrupole Exit 22 V
Focus -11 V
Horizontal Deflect 4 V
Vertical Deflect 1 V
Einzel Focus -15 V
Einzel Horizontal Deflect 3 V


Einzel Vertical Deflect









Table 2-1. Continued.


Repeller 977 V
Pusher 791 V
Doorway 443 V
Long Field Flattener 41.7 V
Short Field Flattener 1 -80 V
Short Field Flattener 2 -80 V
Accelerator 1 228 V
Accelerator 2 -240 V
Accelerator 3 -423 V
Accelerator 4 -672 V
Accelerator 5 -883 V
Flight Tube -4000 V
Detector 2650 V
Threshold 2020


Atmospheric Pressure MALDI Configuration

A simplified diagram of an APMALDI source is shown in Figure 2-4.



Target i\
/ \
I \
lens

I Iris


N2 Laser
Mirror

ND Filter


Figure 2-4. A diagram of the components in a typical atmospheric pressure matrix-
assisted laser desorption/ionization source is shown.

While the arrangement of components varied throughout the design process,

constant to the system were: (1) a 337 nm nitrogen laser (VSL-337-ND-S, Spectra-









Physics, Mountain View, CA, USA), (2) UV enhanced aluminum coated positioning

mirrors (Edmund Industrial Optics, Barrington, NJ, USA), (3) a variable iris (Edmund

Industrial Optics, Barrington, NJ, USA), (4) UV attenuating optics (i.e., neutral density

(ND) filters (Edmund Industrial Optics, Barrington, NJ, USA) or wheels (Reynard

Corporation, San Clemente, CA, USA)), and (5) a fused silica focusing lens (Edmund

Industrial Optics, Barrington, NJ, USA). Two sources were constructed, prototype I and

II. In each case, repetition rate of the laser was monitored using an oscilloscope (TDS

210, Tektronics, Beaverton, OR, USA). Laser power was measured using a pyroelectric

detector (J4-09-030, Molectron Detector, Inc., Santa Clara, CA, USA). Prototype I's

target was a 4 mm diameter stainless steel rod. The newer target, prototype II, is a 2 mm

diameter gold coated post. Target positioning was accomplished in the first prototype by

a stepper driven motorized xyz translational stage (CMA-12CCCL/ESP300; Newport,

Irvine, CA, USA). The reduced size APMALDI source, prototype II, incorporated a

piezoelectric transducer driven xyz stage (8302/IPico Driver, New Focus, San Jose, CA,

USA). Targets were insulated from the positioning devices and held at a voltage ranging

from 0 to 2500 V using an internal power supply from the mass spectrometer.

Table 2-2 lists the potentials of the ion transfer optics and the mass analyzer when

using an APMALDI source.



Table 2-2. Mass spectrometer conditions used to acquire APMALDI spectra.


Nozzle 300 V
Skimmer 75 V
Quadrupole RF 300 V
Quadrupole High 42 V
Quadrupole Low 41.5 V









Table 2-2. Continued.

Quadrupole Exit 20 V
Focus -30 V
Horizontal Deflect 4 V
Vertical Deflect 1 V
Einzel Focus 0 V
Einzel Horizontal Deflect 3 V
Einzel Vertical Deflect 1 V
Repeller 977 V
Pusher 791 V
Doorway 443 V
Long Field Flattener 41.7 V
Short Field Flattener 1 -80 V
Short Field Flattener 2 -80 V
Accelerator 1 228 V
Accelerator 2 -240 V
Accelerator 3 -423 V
Accelerator 4 -672 V
Accelerator 5 -883 V
Flight Tube -4000 V
Detector 2650 V
Threshold 2020 V


Sample Preparation

Solid matrix

The dried-droplet method was used for co-crystallization of the matrix and

analyte.113 Saturated matrix solution was made by dissolving 10 mg of 2,5-

dihydroxybenzoic acid (DHB) (Sigma Aldrich, St. Louis, MO, USA) in 1 mL of 50%

aqueous acetonitrile with 0.05% trifluoroacetic acid (TFA). Reserpine (Sigma Aldrich,

St. Louis, MO, USA) was prepared as a stock solution of 1 nmol-l1 in 50% aqueous

methanol with 0.1% TFA. Peptides were prepared at a concentration of 1 mg-ml-1 in

0.1% aqueous TFA, and diluted as necessary. For crystallization, equal portions of

matrix and analyte were mixed together and -1.5 [tL of resulting solution was placed on









target. The target was placed into position after the solution was dried at ambient

conditions for 20 minutes.

Liquid matrix

Two liquid matrix preparations were used during the ion source optimization stage.

The first matrix used 2,5-dihyroxybenzoic acid (DHB) in a glycerol-solvent solution at a

1:3 mass ratio. The glycerol-solvent solution consisted of 75% glycerol, 15% water, and

10% methanol with 0.1% TFA. The second matrix also used a 1:3 ratio, but of a-cyano-

4-hydroxycinnamic acid (CHCA) (Sigma Aldrich, St. Louis, MO, USA) with

diethanolamine (DEA) (Sigma Aldrich, St. Louis, MO, USA). Each liquid matrix was

sonicated for 10-15 minutes to ensure dissolution. For analysis, 1 il of matrix and 0.5 il

of analyte were mixed on target.



Results and Discussion

Electrospray Evaluation

Using a prototype mass spectrometer, it was important to evaluate instrument

response independent of a new ionization source; therefore, the use of electrospray

ionization (ESI) was incorporated into initial studies. ESI determined a reference point

for ion transmission efficiency, mass resolving power, and ion signal stability.

Figures 2-5 and 2-6 show the total ion counts (TIC) and mass spectra for directly

infused reserpine (m/z 609) and verapamil (m/z 456). For each electrospray spectrum the

resolving power is near 2500, determined by the full width half maximum definition

(Am/m). The percent relative standard deviation for ion counts is -10%, and the mass

accuracy, determined by external calibration and 10 consecutive analysis of each

compound, is between 6 to 8 ppm.









By using a sample introduction rate of 0.5 iL-min1, an extraction frequency of 5

kHz, a total summed transients of 1200, and a sample concentration of 2 ng-LL-1, the

overall efficiency of ion detection can be calculated. The Reserpine mass spectrum

shown in Figure 2-5B provides 1000 counts for the molecular ion (an injection amount of

0.008 ng during the acquisition time), yielding an efficiency of -1 x 10-7 counts per

molecule sampled. While the calculated efficiency is an important parameter, it is not

solely a factor of the instrument; it also includes the electrospray process efficiency.

ESI sensitivity, defined as the slope of the working curve, is determined by both the

efficiency that molecules are converted to gas-phase ions and the efficiency that the

formed ions are transferred through the mass spectrometer and detected.116 The fraction

of ions analyzed then depends upon the transfer optics and the mass analyzer. Absolute

efficiency of the ESI process is unknown; however, the literature generally agrees that the

limiting factor in sensitivity is ion transmission, not formation.116 Thus, as an

instrumental parameter, the overall efficiency obtained with ESI can be considered a

maximum. With optimized ion transmission into the inlet, and estimate for signal

intensities can be based upon ion formation efficiencies.

APMALDI Source

Prototype I

The original configuration for the APMALDI source was designed to allow

maximum adjustment for the optical components; therefore, standard optical elements

and hardware were used for the assembly. Figure 2-7 is a photograph of the prototype I

source. Inset in the figure is a magnified view of the target assembly.










Reserpine


400


500


1000 1100


Figure 2-5. Plots showing the A) total ion count chromatogram and B) a mass spectrum
for the analysis of reserpine. Inset in the figure is a ball and stick image of the
molecule analyzed.


1200
1000
800
600
400
200
0


100


200


300


Time (s)


600


[M+H]+


1200
1000
800

600
400
200
0 -
500


600


700


800


900


m/z


1200


-----e~L---- ---------- ---- -------- -r ---- ------- ------- ----- ---


"WiMULL NO 1116HM-Y1 W'"ILAIIW-JOA~llW











Verapamil


3500
3000
2500
2000
1500
1000
500


100


200


300


400


500


600


Time (s)


1200

1000

800

600


400

200

300
300


[M+H]+


500


700


900


1100


m/z



Figure 2-6. Plots showing the A) total ion count chromatogram and B) a mass spectrum
for the analysis ofverapamil. Inset in the figure is a ball and stick image of
the molecule analyzed.


~c~









The 337 nm nitrogen laser outputs a rectangular beam area of approximately 5 mm

by 7 mm with a pulse width of 4 ns. In this arrangement the laser beam is focused to a

spot size on target of -250 rm by -300 rm in diameter (elliptical shaped). The target is

held at -45 degrees from the normal of the laser beam. The original electrospray curtain

gas cover was modified by reducing a 22 mm diameter section of the piece to 1 mm in

thickness. In the electrospray mode, the original and modified covers produced identical

results; however, the benefit for APMALDI was that the new cover allowed the target to

move within 2.5 mm of the orifice.

Solid matrix. When ion production is not consistent, source optimization is

difficult. Since the oa-TOF is run asynchronously with the laser pulse, single shot spectra

(as done typically with vacuum MALDI on a linear TOF) produce drastically different

ion intensities, negating any attempt at parametric studies.

At this point in ion source development, a continuous ion signal was needed. In a

preliminary attempt, the laser repetition rate was increased to 10 Hz to produce a quasi-

continuous ion beam, but increased laser frequency rapidly depleted the solid sample.

Figure 2-8 is a mass spectrum of solid matrix (DHB) obtained using the prototype I.

While the quasi-molecular ion [M+H] is visible, DHB clusters are seen throughout the

m/z range.

With the laser operating at 10 Hz and the xyz motorized stage translated

continuously (rate of 0.5 mm per minute), solid matrices still produced erratic ion

production. Figure 2-9 shows total ion count (TIC) traces for the solid and liquid

matrices. The lower trace demonstrates the ion signal reproducibility obtained with solid

matrices. The drastic variations in the ion trace occur due to heterogeneity in the matrix









crystals. The idea of a liquid matrix was appealing during this stage of development;

however, to accommodate solution droplets, the target angle needed to be altered.


Figure 2-7. A photograph of the first constructed APMALDI source, prototype I. Inset in
the figure is a magnified view of the target assembly.










1200

CH30 N
[M+H]+ H H
1000- H- H o ,
[2DHB-2H20+H]+ H oH ,C-
C 0 CHC

S800m/z 397
U, 800



" 600
N

E [3DHB-3H20+H]
o 400 [4DHB-4H20+H]+

[5DHB-5H20+H]

200 [M-CoH,1C+H] [6DHB-6H20+H]+
[DHB-H2O+H] [7DHB-7H20+H]


0 200 400 600 800 1000 1200

m/z


Figure 2-8. A mass spectrum of reserpine analyzed in a solid DHB matrix is shown.
Analysis was done using the prototype I APMALDI source.

Liquid matrix. A photograph of the target assembly constructed for liquid

matrices is shown in Figure 2-10. Inset in is a computer aided drawing (CAD)

(Solidworks, Concord, MA, USA) of the target showing its relative position and the laser

angle used for desorption. Additionally, a three dimensional depiction of the target

assembly and its orientation to the orifice is shown using a executable output file from

the CAD program.

Object 2-1. A file showing the target alteration for the liquid matrix (1.8 mb,
PrototypeI.exe, repeating play file).










100
0 OH
90-
OH
80-

70 HO 2 5 Dhydroxybenzoic acid

6 60

S50- Liquid matrx

- 40

30

20- Solid matrix

10


0 100 200 300 400 500
Time (s)


Figure 2-9. Plot of the total ion count chromatograms for solid and liquid matrices. DHB
was used as the absorbing chromophore with reserpine as the analyte.

Figure 2-11 shows a liquid matrix mass spectrum collected using the new target

holder. The spectrum is an improvement over the solid matrix analysis (i.e., reduction of

matrix clusters). While improvements in solid matrix preparations can also lower the

background, the important feature of the liquid matrix is the continuous ion production,

as seen in Figure 2-9. In the TIC trace, as the laser is operated at 10 Hz, the liquid matrix

sample stage is kept in a fixed position. This provided extended ion production, enabling

interface optimization.

Atmospheric pressure sampling. Critical in atmospheric pressure (AP) interfacing is

cluster prevention; therefore, during source optimization the curtain gas cover, gas mass

flow rates, and interface potentials were examined as they related to maximum ion signal









production. Additionally, moving ions at AP is difficult due to the number of collisions

occurring. Unlike the reduced pressure flight tube, the mean free path of an ion at

atmospheric pressure is on the order of nanometers.


Figure 2-10. A photograph of the altered target assembly for liquid matrix analysis. Inset
is a computer aided drawing of the target showing the laser angle.










1200



1000-



800-



600-



400-



200-



0


[M+H]+


CHaO !OC
H H
H I OCH,
0 H 3H "OCH,


[M-CloHO1,+H]+


. 1 ..


[2DHB-2H20+H]
S I


200


400


600

m/z


800


1000


1200


Figure 2-11. Mass spectrum of reserpine in a DHB liquid matrix taken using the modified
target assembly is shown.




Mean free path can be expressed by the following equation:

kT (2-1)



where the mean free path (k) is related to the collisional cross section (C), pressure (p),

Boltzman constant (k), and temperature (T).117 Since effective transmission is important

in improving ion signals, the distance between the target and the orifice was reduced in

an attempt to increase ion signals. To minimize the target-orifice distance, the curtain gas

cover was removed. Figure 2-12 is a photograph of the source and target assembly with





..L' IL I








the curtain gas cover removed. This image can be compared with Figure 2-10 where the

curtain gas cover is in place.

I % 0', 0 9 6A1*61%* *M
I** *pf a


Figure 2-12. A photograph of the source and target assembly with the curtain gas cover
removed.

No ion signal differences were visible with the removal of the curtain gas cover

alone; however, when the target was positioned closer to the orifice the ion signal

intensities increased -30%. Figures 2-13 A and B show mass spectra obtained at the

different target-orifice distances of A) 1.5 mm and B) 2 mm.

















750




C 500




250


500 1000 1500
m/z


2000


500 1000 1500
m/z


2500 3000


2500 3000


Figure 2-13. Mass spectra obtained with the target placed at a distance of A) 1.5 mm
from the orifice and B) 2 mm from the orifice.

Moving the target closer than 1.5 mm from the nozzle resulted in high voltage arcs,


and reductions in the voltages reduced ions signals to levels below that obtained at larger


distances. Interestingly, a 25% decrease in target-orifice distance allowed a 30% increase


in ion signals; however, this does not suggest a direct relationship.


A
[M+H]+ 1.5 mm






J l ,'


n-i. -~ _________________________________


1000


750




c 500




250


B
2 mm



[M+H]+











,. l ,


04 U -.''' ~~'









Prototype II

The second iteration of the APMALDI source minimized optical component

positioning and reduced the overall size. Figure 2-14 is a photograph of the second

APMALDI prototype. Inset in the figure is a new target assembly that positioned the

translational stage away from the orifice axis. The impetus for moving the translational

stage away from the orifice will be described in Chapter 6. Additionally, a three

dimensional depiction of the prototype II source is shown in Object 2-2.

Object 2-2. A file showing the prototype II source (1.8 mb, PrototypelI.exe, repeating
play file).


Figure 2-14. A photograph of the second APMALDI source, prototype II. Inset in the
figure is a new target assembly that positioned the translational stage away
from the orifice axis.









The prototype II APMALDI source uses a smaller diameter (2 mm) target with

rounded edges. The edges reduced the possibility of arcing from the target to nozzle.

The target is also coated with gold to minimize sample carryover. Target positioning is

done using piezoelectric transducer driven motors. The smaller piezoelectric transducers

not only diminished the source's overall size, but also minimized the controller, which fit

inside the mass spectrometer housing. An additional advantage of the modified source

was safety. The target assembly completed a closed loop circuit to initiate all interface

voltages. Accidental shocks were reduced. With miniaturized optical components (New

Focus, San Jose, CA, USA), the complete source (sans laser) could be constructed inside

an enclosure attached to the mass spectrometer-a key benefit for commercial

compatibility.

Adjusting Interface Parameters

Initial measurements taken with the curtain gas flow indicated that higher flows

yielded larger analyte ion intensities and reduced background. The original device used a

limited 1 L-min-1 mass flow controller. To increase the range, a 5 L-min1 mass flow

controller was added to the system. Figure 2-15 shows mass spectra as the counter-

current gas flow is set at 0, 1, and 5 L-min', respectively.

In each case the analyte signal, angiotensin I (m/z 1296.68), increased while the

background, DEA dimer (m/z 211), was reduced. Additionally, the ratio of sodium

adduct [M+Na] to protonated molecular ion [M+H] was minimized with a 5 L-min-1 gas

flow. Considering the fundamentals of the cluster prevention mechanism using a

counter-current gas flow, it seems reasonable that increased gas flows (counter-current)

should provide analyte signal increases through a reduction in clusters.








56



1500 1500
0 L/min A 1 L/min B
1200 1200


. 900- 900
C C
S600 600
[M+H]+
[M+H]+
300 [DEA+H] 300 [DEAH][M+Na]
500 [DEA+H] [

0 500 1000 1500 2000 0 500 1000 1500 2000
m/z m/z


1500
5 L/min C
1200 [M+H]


S900


600

[M+Na]*
300
[DEA+H]r
0I "-----1 ..
0 500 1000 1500 2000
m/z


Figure 2-15. Three mass spectra taken using gas flow rates of A) 0 L-min1, B) 1 L-min-1,
and C) 5 L-min-1. Angiotensin II was used as the analyte in a CHCA liquid
matrix.


The original literature for APMALDI saw a different effect for gas flow, indicating


concurrent flows provided ion signal increases.104,106,114 In original APMALDI


orientations an angled probe tip was used for the sample and a stream of nitrogen gas


directed at the nozzle entrained the ions. Only recently has literature pointed to counter-


current gas flows increasing ion signals.115


The effect of temperature on the prevention of water clusters for supersonic jet


expansion is known; thus, temperature was empirically examined.102 A constant 1000 C


provided maximum ion signals without arcing. Higher temperatures provided no signal









increases or background decreases, yet more frequent arcs between the target and the

nozzle occurred.

With an optimized target-orifice distance, target voltage was examined as it related

to analyte ion yield. Although target voltage can be varied independently, it does not

function independently for ion transmission. Instead, it is related to nozzle voltage, the

voltage applied to the orifice. Figure 2-16 shows two diagrams of the mass spectrometer

interface.



B j







itrogen gas flow Orifice = 254 pm 4


Target Skimmer





35.5 mm
irget distance = 1.5 mm
2 kV 300 V 75 V

Figure 2-16. An illustration of the mass spectrometer interface. The two images show A)
the gas orifices and mass spectrometer inlet, and B) the arrangement of the
target, nozzle, and skimmer.

Together the target and nozzle form the electric field that transports ions from

atmospheric pressure into the spectrometer. Figure 2-17 shows the interrelated functions

of target voltage, nozzle voltage, and analyte (bradykinin fragment 1-7 m/z 756.4) ion

yield.















3000


2500


2000


1500


1000. ,
2000

500 1500 IQ

0 1000 \@
3500

300 500
250

S ge (V) 150


Figure 2-17. A three dimensional plot showing the relationship of target and nozzle
voltage to analyte ion yields. The skimmer was maintained at 75 V.
Bradykinin fragment 1-7 (m/z 756.4) was the analyte in a CHCA liquid
matrix.

Two events are demonstrated in Figure 2-17. First, increased ion yields occur at

increased electrostatic fields. The maximum signals occur near 1100 V-mm-1 (1700 V at

a distance of 1.5 mm); however, the signals are not maintained as the target and nozzle

voltages are lowered. Second, the potential for the skimmer is 75 V; therefore, a

reduction in the nozzle voltage reduces the field between the nozzle and skimmer.













2500


2000









12
S1500
S ^ -------------













00 80 45
5., 1000 .







500400







60 250 300
141500


-100 100






Figure 2-18. A three dimensional plot showing the relationship of nozzle and skimmer
voltages to analyte ion signal intensity. The target was maintained at 2 kV.
Bradykinin fragment 1-7 (m/z 756.4) is used as the analyte ion in a CHCA
liquid matrix.

Figure 2-18 was produced to investigate the ion intensity relationship between the

nozzle and skimmer. For Figure 2-18, a broader range of electric fields, versus Figure 2-

17, could be applied without significantly reducing the ion signals. In part, this may be

due to reduced pressures between the nozzle and skimmer.

Mass spectrometer conditions. In a supersonic beam the energy of the ions is

mass dependent due the ions being at constant velocity. When using an oa-TOFMS, a









mass dependent drift occurs in ion trajectories for the flight tube axis. Ions do not

traverse the flight tube completely perpendicular to the pulser; some flight tubes are

offset to account for the trajectory angle. However, since each mass requires a slight

change in trajectory, methods must be incorporated to allow for a large range of different

masses to reach the detector. The methods include using a large detector area to cover

the trajectory range, providing deflection plates for specific masses, and adding energy to

the supersonic beam to minimize the initial energy spreads (a larger total energy relative

to the initial kinetic energies). An alternative approach is to use a collisional focusing

RF-only multiple device containing an inert gas at elevated pressures (0.01 1 Torr).9-10

The collisions with the gas reduce the average ion energy. Also, the multi-pole device

focuses the ions to a beam as they approach the thermal energy of the gas.119-120 Thus,

the beam leaving the ion guide has a smaller spatial spread in the axis of the flight tube,

and these properties are almost independent of the original parameters of the ion beam

delivered by the source (i.e., no memory of spatial or kinetic distributions from the ion

formation processes).89

Further optimization of the mass spectrometer conditions (pulser voltages, Einzel

lens voltages, etc.) was undertaken, but showed little effect. The optimization of the

parameters for ESI and APMALDI (besides the interface and initial ion transmission

parameters) yielded similar results. The mass spectrometer voltages remain constant

primarily due the sampling method of the jet expansion.117 While the conditions are not

directly related to the jet expansion, they are indirectly related due to the use of the

collisional focusing in an RF-only quadrupole.









Conclusions

This chapter demonstrates the initial instrument characterization, using an ESI

source, and the construction of an APMALDI source. Using an oa-TOFMS, the

optimization of interface parameters (i.e., target distance, gas flow, target voltage, nozzle

voltage, and skimmer voltage) must be accomplished to maximize APMALDI ion

collection and transmission. An added benefit to the use of an RF-only quadrupole,

besides collisional focusing, is the reduction in ion source memory, easing spectrometer

parameter adjustments. The APMALDI source designed and implemented presents an

ion source that would be easily interchangeable with a common ESI source configured

mass spectrometer. The adjusted parameters and their relationship to ion yields

demonstrates the necessity of optimizing atmospheric pressure transmission.

Additionally, liquid matrices were introduced as the first analytical advantage for

APMALDI. Further evidence for this is demonstrated in upcoming chapters.














CHAPTER 3
LIQUID SUPPORTS FOR ULTRAVIOLET ATMOSPHERIC PRESSURE MATRIX-
ASSISTED LASER DESORPTION/IONIZATION

Introduction

Matrix-assisted laser desorption/ionization (MALDI) has seen widespread use in

bioanalytical analysis.111 For analysis of large intact biomolecules, MALDI requires a

suitable matrix to absorb energy and transfer it to the analyte. Many types of matrices

exist, yet the most widely accepted today is some form of solid, crystalline structure that

acts as an analyte host. This solid, low volatility matrix allows convenient application in

the low pressure environment of a mass spectrometer ion source; however, the rigid

lattice matrix presents a heterogeneous sample surface for successive laser pulses. As the

crystal surface is ablated, analyte ion signals fluctuate due to the non-uniform sample

surface.121 Enhanced homogeneity of the surface through sample preparation assists in

providing more reproducible MALDI analyses.122-24 Despite the numerous sample

preparation methods reported, sample heterogeneity remains an issue.121,125-127

A liquid matrix, with its self-renewing surface, eliminates the sample heterogeneity

problem associated with solid matrices. Liquid sampling systems have found prior use in

mass spectrometry to combat signal irregularities and to provide increased signal lifetime.

Fast atom bombardment (FAB) and liquid secondary ion mass spectrometry (LSIMS)

demonstrated the use of viscous liquids as an effective sample surface.128-129 The process

of energy transfer differs in these two methods, but the surface replenishment principle is









similar. Drawing on that success, laser desorption methods have also incorporated the

use of liquid supports.34

A number of MALDI liquid supports have been studied, including neat liquids,

particle suspensions, and chemical doping. Neat liquids provide direct absorption of the

laser and limit matrix preparation time. Unfortunately, relatively few vacuum stable

liquids provide adequate UV absorption.130-133 The addition of absorbing particles to a

vacuum stable liquid, a particle suspension matrix, provides a low volatility medium that

absorbs UV wavelengths.134-137 These particle suspension matrices allow desorption and

ionization, although the mechanism for desorption is not typical of a solid MALDI

matrix.45 The particle suspension matrix has been regarded to induce a thermal event,

whereby rapid heating at the particle surface allows thermal desorption of analytes.134,136-
137 Chemically doped liquid matrices may be more analogous to solid MALDI systems

with their use of energy absorbing molecules.138-139 A variety of absorbing molecules

have been used in binary mixtures with some success.132,138,140 Wang et al. developed

vacuum stable chemically doped liquid matrices by using typical solid MALDI

chromophores, 2,5-dihydroxybenzoic acid (DHB) and a-cyano-4-hydroxycinnaminic

acid (CHCA), in viscous liquids such as glycerol and diethanolamine (DEA).139 In some

cases, a solubilizing agent was added to the mixture to ensure chromophore dissolution.

Liquid matrices have provided excellent shot-to-shot reproducibility and long-term

analyte signal stability; however, the liquid systems have been evaluated in a vacuum ion

source-necessitating a low volatility medium.132'138 Liquid matrices placed in a vacuum

environment have encountered problems with source contamination and high backing

pressures (5x10-6 Torr), limiting their incorporation for routine analysis.132,137 The









development of atmospheric pressure (AP) MALDI provides new opportunities for liquid

matrices under conditions where sample volatility need not be as restricting.104-105'109 An

APMALDI source provides good limits of detection and has been suggested as a softer

ionization method due to increased collisional cooling.114 With the MALDI ion source

operating in an open ambient environment, vacuum stable matrices are not necessary.

To expand sample analysis opportunities, while providing the benefits of traditional

MALDI chromophores, we have explored suitable liquid matrices for UV APMALDI.141

Glycerol-based liquid matrices, which absorb in the IR, have become common for use

with IR lasers at 2.94 rm and 10.6 im.48'114,142-143 However, due to cost and availability

issues, IR laser systems are less commonly used for MALDI applications. The UV

nitrogen laser (337 nm)-simple to use, relatively inexpensive, and readily available in

many laboratories-has become the most widely used MALDI laser source.48'142

Our experimental goals focused on developing a UV-compatible liquid matrix by

doping a typical MALDI chromophore, a-cyano-4-hydroxycinnamic acid (CHCA), into a

liquid medium. The liquid support comprises a solvent liquid for analyte solubility and a

viscous component for signal lifetime. Formulation of a UV absorbing liquid matrix for

use at AP presents unique problems and advantages. As noted above, Wang et. al

demonstrated similar studies for the formulation of chemically doped matrices optimized

for a vacuum ion source.139 Out of the vacuum, matrices are not limited by vapor

pressure or source contamination; however, desorption and ionization at AP must be

characterized through alterations in the liquid systems. We show an example of an AP

liquid matrix that provides an effective avenue for UV APMALDI analysis. In this

chapter, we report on the use of suitable liquid matrices for UV APMALDI, showing ease









of use, representative spectra, and promising quantitation. The parameters studied

include chromophore concentration, liquid support variations, and quantitation capability.

We believe this approach offers advantages that complement current MALDI methods.



Experimental Methods

Atmospheric Pressure MALDI Source

The mass spectrometer is an orthogonal-acceleration TOFMS (LECO Corporation,

St. Joseph, MI, USA), which is described in detail in Chapter 2. The APMALDI ion

source used a 337 nm nitrogen laser (VSL-337-ND-S, Spectra-Physics, Mountain View,

CA, USA), focused by a fused silica lens, to irradiate the sample on a gold coated target,

2 mm in diameter. The laser spot was -250 [tm by -300 [tm in elliptical diameter and

yielded -60 to 80 PJ per pulse. The target, onto which the sample and matrix were

deposited, was positioned relative to the MS orifice using a motorized xyz translational

stage (8302/IPico Driver, New Focus, San Jose, CA, USA). Held at 2 kV, the target was

on-axis -1.5 mm from and -1 mm below the 254 [tm orifice, which was maintained at

300 V. Ions from the matrix/sample solution were transferred into the spectrometer using

a counter-current gas flow interface. The nitrogen flow was set to 5 L-min-1 and heated to

1000 C.

Solution Preparation

The matrix was prepared by mixing CHCA (Sigma-Aldrich Corp., St. Louis, MO,

USA) with the liquid support made from a solvent liquid and a viscous component. The

solvent liquids used were ethanol, acetonitrile, acetone, and water (Fisher Scientific, Fair

Lawn, NJ, USA). Aqueous solutions of trifluoroacetic acid (TFA) (Sigma-Aldrich Corp.,

St. Louis, MO, USA) were also used as test solvents for the liquid matrix. The viscous









component was diethanolamine (DEA) (Sigma-Aldrich Corp., St. Louis, MO, USA).

Each matrix preparation was sonicated and vortexed to ensure dissolution. Peptides were

prepared in aqueous acetonitrile with 0.1% TFA, unless otherwise stated. Sample

analysis was conducted by spotting 0.5 gL of matrix onto 0.5 [tL of analyte solution.



Results and Discussion

Liquid Matrices

Current mass spectrometers dedicated to electrospray ionization (e.g., oa-TOF) are

directly amenable to APMALDI, yet the advantages of using non-vacuum compatible

matrices have not been broadly investigated.109,143-144 The liquid matrix used in these

studies comprises a chromophore, a support liquid, and a solvent liquid. The

chromophore, CHCA, absorbs and transfers energy to the analyte in a controlled

manner.37 The support liquid, DEA, is used as a viscous component for sample

longevity; however, it also acts as a solubilizing agent for CHCA.136,139 The solvent

liquid is used for reducing viscosity, allowing for enhanced signal intensity and an

increase in analyte solubility.

Chromophore Concentration

In solid sample MALDI, CHCA crystals are embedded with analyte, leaving the

crystalline matrix to serve directly as the chromophore.37 In this mode various matrix-to-

analyte molar ratios are possible, providing different degrees of analyte isolation and

energy transfer.145 Matrix-to-analyte ratios are believed to play a role in effective analyte

isolation.45 As the analyte molecules become more isolated from one another, efficient

soft desorption and ionization occurs.









In the liquid system, the matrix-to-analyte ratio is not based only on CHCA

amounts. The liquid matrix differs in that it contains water, ethanol, and diethanolamine

in large quantities to isolate the analyte without the addition of the chromophore. Thus,

analyte isolation within the support liquid can be maintained while chromophore

concentration is studied. To evaluate chromophore concentration dependence, CHCA

amounts doped into the liquid support were altered while monitoring analyte ion

intensity. The liquid support consisted of a solvent liquid, 50% aqueous ethanol, and a

viscous component, diethanolamine. Figure 3-1 shows the analyte and matrix

background mass spectrometry ion counts as the chromophore concentration is increased.

The protonated analyte, angiotensin II (m/z 1046.5), and the matrix background,

total ion count (TIC) below m/z 300, are plotted against CHCA concentrations. At

concentrations below 100 mM, insufficient laser energy is absorbed to effect desorption

and measurable ionization of the analyte. As the concentration increases, both the

analyte and matrix signals rapidly increase. Up to approximately 800 mM of CHCA,

there is a direct relationship between analyte ion intensity and the amount of added

chromophore; however, further additions of absorber actually lower the analyte signal.

As the chromophore concentration is altered the absorption characteristics of the

matrix are changed. The CHCA allows the coupling of the laser energy to the solution,

so with increasing chromophore concentration, the amount of laser energy absorbed in a

finite volume on the surface of the solution also increases.










12000 1400
a-cyano-4-hydroxycinnamic acid (CHCA) Background < 300 m/z
@ 337 nm 24,200 L mol-1 cm-1
--0-- [M+H]+
1200
10000 1200

-J
1000
:> 8000

80 800


o
6000

P 600 '
o C

S4000
,. -400


2000 1 200



0 0
0 200 400 600 800 1000 1200 1400

CHCA Concentration (mM)

Figure 3-1. The plot shows analyte ion and matrix background intensity as a function of
CHCA concentration in the liquid matrix. The matrix background is the total
ion count for all species below m/z 300. Fifty picomoles of angiotensin II was
spotted on target to monitor analyte signal intensities. Chromophore
concentration changes were done using one liquid matrix support, 50%
aqueous ethanol mixed with an equal part DEA.



The Beer-Lambert law indicates that the laser intensity transmitted decays

exponentially with distance and a constant related to absorber concentration.145

kb (3-1)
-e
(O









Where P is incident laser flux, (o is transmitted laser flux, b is the distance from

the solution surface to the measurement of the transmitted power, and k is dependent

upon concentration and molar absorptivity (E).

k c (3-2)
k-
0.434

Given a value of s = 24,200 L mol-1 cm-1 for CHCA, we can estimate the

penetration of the laser into the optically thick solution.139 Using the 1/e definition of

laser penetration depth, we obtain a distance of -300 nm for the 630 mM concentration.

This concentration provided maximum analyte ion signal and was used in the studies

presented below. The concentration range studied would suggest laser penetration depths

from 100 nm to 30 im.

The penetration depth may provide some rationale for the trend observed in Figure

3-1. As the chromophore concentration rises, the effective penetration volume of the

laser is lowered, thus reducing the number of analyte molecules affected by each laser

pulse. For a laser spot size of -300 rim, an estimate for the effective sample volume can

be determined (saturation effects for the absorbers were not considered due to the low

laser powers used). Using the 630 mM CHCA concentration, the laser penetration

volume element is estimated at -1.5 x 104 am3, within which there would be 3 x 1014

water molecules, 2 x 1013 ethanol molecules, 2 x 1013 DEA molecules, and 3 x 1012

CHCA molecules. Considering 5 picomoles of angiotensin II loaded on target, the

volume element of the laser would contain -75 attomoles of analyte, 4.5 x 107

angiotensin II molecules.

Figure 3-2 shows a mass spectrum collected using the 630 mM CHCA matrix with

5 picomoles of angiotensin II loaded on target. The major analyte peak is the [M+H]+,







70


with some sodium and potassium adducts also present in the spectrum. The insert, Figure

3-2A, is an expanded view of the protonated analyte peak showing a resolution of -2500.

The analyte also forms an adduct with DEA, [M+DEA+H]+, at m/z 1152.5. The

matrix background shows peaks for protonated DEA (m/z 106) and the protonated DEA

dimer (m/z 211). Other unidentified peaks also reside below m/z 300 at low intensity

levels.


1000




800 -1


600

cn

400




200


0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
m/z

Figure 3-2. Mass spectrum of 5 picomoles of angiotensin II analyzed using an optimized
CHCA liquid matrix. The spectrum is a 5 minute summation with the laser
operating at 20 Hz. The liquid matrix contained 630 mM CHCA and a 50%
aqueous ethanol solution mixed with an equal part DEA. Figure 3A is a
scaled view of the analyte peak and adducts.

To further examine chromophore concentration adjustments, a different

chromophore was incorporated into the matrix. Studying a second chromophore may


A [M+H]+







[M+Na]+
[M+K]1
[M+K] [M+DEA+H]+


1000 1050 1100 1150 1200


A



~II"










determine if the absorption required for desorption and ionization is independent of the

absorber. Each MALDI chromophore has a unique absorption distribution as shown in

Figure 3-3; however, all provide useful results when used with the proper analyte type

and with the correct matrix-to-analyte ratio.


UV absorption of Matrices
90000
340nm
80000 CHCA

70000

> 60000

S50000

40000 323nm
303nm SA
30000 HPA

20000
332n DHB
10000 3

0
200 250 300 350 400 450 500
Wavelength (nm)

Figure 3-3. An illustration of the UV-Vis absorption spectra collected for common
MALDI matrices is shown. Adapted from reference 147.

The MALDI chromophore 2,5-dihydroxybenzoic acid (DHB) is considered a general UV

matrix useful for a variety of analyses. As a comparison to CHCA results, DHB

concentration studies were conducted. Figure 3-4 shows the analyte ion signals

(substance P m/z 1347) versus DHB concentration.

With a lower molar absorptivity at 337 nm (3800 L mol-1 cm-1), higher

concentrations of chromophore (DHB) were required for maximum analyte signals.

Further increases in the DHB concentration could not be studied due to solubility







72


difficulties. A comparison of DHB to CHCA concentration at maximum analyte signals

shows a ratio of -6.8:1 (4300 mM/630 mM).

1200
2,5-Dihydroxybenzoic acid (DHB)
@ 337 nm 3800
1000


S800 a -


600


400


200 ..


0
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Concentration (mMI

Figure 3-4. A plot of analyte ion intensity as a function of DHB concentration is shown.
Fifty picomoles of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-
Met) was deposited on target. Chromophore concentration changes were done
using one liquid matrix support, 50% aqueous acetonitrile with an equal part
glycerol.

This ratio is equivalent to the ratio of molar absorptivities 24,200/3,800 -6.4:1. These

results may point to an optimal value for absorption required to yield analytical results;

however, studies including other matrices must be compared.

Support Liquid Variations

The non-absorbing components in the liquid matrix are the solvent liquid and the

viscous component.139 DEA has been used as a viscous component to enable liquid

analysis in vacuum MALDI; however, APMALDI allows liquid matrix volatility

constraints to be reduced.139 While the addition of DEA to a volatile solvent limits

evaporation, enabling increased sample lifetimes, the ratio of viscous component to









solvent liquid requires investigation. Using the CHCA concentration that provided the

largest analyte ion signal (-630mM), different ratios of solvent liquid to DEA were

examined. The solvent liquid used was a 50% aqueous ethanol solution. As the viscous

fraction, DEA, was decreased, the solvent liquid was increased to maintain a 1 taL

volume, with a fixed chromophore concentration.

Reducing the DEA fraction limits the liquid sample lifetime, and thereby the

available time for sample analysis; however, the total viscosity of the liquid matrix also

plays a role in analyte desorption. Increased viscosity represents strengthened

intermolecular forces. In this case, more DEA in the mixture allows additional hydrogen

bonding, thereby requiring more energy for desorption to occur. Desorbed molecules

require additional energy to overcome the increased intermolecular forces, making

desorption from the liquid phase more difficult. Figure 3-5 shows the analyte signal

intensity as the percentage of DEA is increased in the liquid matrix.

Above 50% DEA, analyte signal intensities decrease as viscosity increases, but the

signal lifetime is prolonged. The typical analytical lifetime for a 50% DEA liquid matrix

during laser desorption is 30 minutes. While analysis can be completed in 1-5 minutes,

increased lifetimes allow both source optimization and larger summation times for signal-

to-noise enhancements. The 25% and 75% solutions provided signals for -10 and -60

minutes respectively. The 0% DEA mixture has a limited lifetime, < 1 minute, and

provided low analyte signals with large variations. Also, without diethanolamine added

to the matrix, the water and ethanol were not effective in solubilizing the CHCA, so the

mixture was not homogeneous. The 100% DEA mixture was also a heterogeneous

mixture due to the difficultly in mixing such a viscous solution.







74




250



200



150



= 100



50




0% 25% 50% 75% 100%
% DEA in liquid matrix


Figure 3-5. A chart of analyte intensity versus the percentage of DEA in the liquid matrix
is shown. The signals represent 1 minute summed mass spectrometry signals.
Five picomoles of angiotensin II was placed on target for analysis.

From measured sample lifetimes, liquid removal rates can be estimated. For the

50% DEA mixture, -14 picoliters per laser pulse is estimated based on the amount of

liquid spotted (1 gL) and the amount remaining after analysis (-0.5 gL). Analyte

removal rates can be determined using the analyte signal lifetime and the amount loaded.

A 5 picomole sample lasts -36,000 laser shots, yielding a single pulse analyte removal

rate in the attomole range. While removal rates are influenced by liquid matrix

composition, the summation of analyte signals for longer periods allows enhancements of

signal-to-noise.148

Composition of the solvent liquid is also important for the liquid matrix. To

determine the effect that solvents have on analyte signal intensities, we examined










common solvents. Figure 3-6 demonstrates the effect solvent variations have on analyte

ion signal using a 50% DEA liquid matrix.


1000




750




500




250




0
Water Ethanol Ethanol/Water 0.1% TFA Acetonitrile Acetone


Figure 3-6. A chart of analyte intensity versus solvent liquid used in the liquid matrix.
Each column is the solvent added to a 50% DEA mixture. The signals
represent 1 minute summed mass spectrometry signals. Fifty picomoles of
angiotensin II was placed on target for analysis.

The CHCA concentration was kept constant at -630 mM. The analyte signals for

the solvent studied provided consistency in intensity and reproducibility. Spectral

backgrounds for the solvents were also comparable.

While a limited variety of solvents was tested with the liquid matrix, the results

demonstrate a flexible range over which the matrix can provide useful results. A select

solvent may be necessary for specific analyte solubility. Also, considering typical

reverse phase liquid chromatography (LC) solvents, alternative matrix solvents could

prove useful for online LC/APMALDI.









Solids versus Liquid Matrices

For liquid matrices to be useful in analytical analysis they must provide analyte

signals comparable to solid matrices. To examine the comparison, a study was conducted

using the dried-droplet solid matrix preparation and the optimized liquid matrix

preparation.37 For the solid matrix analysis 1 mL of 50% ACN in 0.05% TFA solution

was mixed with 10 mg of CHCA matrix. Next, 10 [tL of analyte sample was mixed with

an equal volume of matrix solution. One microliter of the mixture was spotted onto the

target and allowed to dry for 20 minutes. Bradykinin fragement 1-7 (m/z 757.4), human

angiotensin II (m/z 1046.5), human Adrenocorticotropic (ACTH) fragment 18-39 (m/z

2465.2), and synthetic peptide P14R (m/z 1533.8) were dissolved in aqueous 0.1% TFA

(Sigma-Aldrich Corp., St. Louis, MO, USA). The same peptide solutions were used for

liquid matrix analysis. The liquid matrix consisted of 630 mM concentration of CHCA

with a support liquid of 50% DEA and 50% aqueous ethanol. The deposited liquid

matrix consisted of 0.5 [tL of matrix solution placed on top of 0.5 [tL of analyte solution

to ensure mixing. For both solid and liquid analysis, total peptide placed on target was 25

picomoles.

Figures 3-7 to 3-10 show MALDI mass spectra for peptide analytes using either

solid and liquid matrix preparations. All mass spectrometer and source conditions were

kept constant. Figures 3-7 and 3-8 show bradykinin fragment 1-7and angiotensin II

analyzed with both solid and liquid matrices.







77



3000

Bradykinin 1-7 Solid
2500


2000


| 1500
3i

1000


500 [M+H]+


0
3000


2500 [M+H] Liquid


2000


c 1500
c-


1000

[M+Na]

500 [M+K]* [M+DEA+H]*


04 ..-L I k '
500 600 700 800 900 1000
m/z


Figure 3-7. Mass spectra of bradykinin fragment 1-7 comparing solid and liquid matrix
preparations. Twenty-five picomoles of analyte was deposited on target, and
each spectrum was the summation of 100 individual spectra.










2000

Angiotensin II Solid


1500




C 1000
C


[M+H]+
500





2000

[M+H]' Liquid


1500




C 1000
C




500
[M+Na]+

[M+K]+


0 ______iL___-
500 750 1000 1250 1500
m/z


Figure 3-8. Mass spectra of angiotensin I comparing solid and liquid matrix preparations.
Twenty-five picomoles of analyte was deposited on target, and each spectrum
was the summation of 100 individual spectra.









In both Figures 3-7 and 3-8, the protonated molecule signals can be seen in the

solid and liquid matrix formulations. The liquid matrix differs in signal intensity and

adduct formation. Though the liquid matrix presents larger [M+H] signals, no fluence

adjustments were made to optimize solid matrix analysis; therefore, conclusions cannot

be drawn on the limits of detection of liquid versus solid matrices. However, what can be

considered in Figures 3-7 and 3-8 is the appearance of alkali metal and DEA adducts.

While DEA is not available in the solid matrix formulation, sodium and potassium are

present. The formation of adducts is a known phenomenon for APMALDI.114 Adducts

in the liquid matrix suggest a softer desorption/ionization event. Collisional cooling at

AP is thought to provide the stabilization for adducts to remain intact. Softer ionization

modes can provide benefits when examining non-covalent interactions or analyzing

fragile compounds (e.g., deoxyribonucleic acids). Additionally, instrumental and sample

preparation methods are available to reduce analyte adducts (e.g., ultra-clean targets,

high-grade reagents, larger declustering voltages between nozzle and skimmer).

Figure 3-9 shows ACTH fragment 18-39 analyzed with both solid and liquid

matrices. In Figure 3-9 no analyte signal differences were present between the solid and

liquid matrix preparations. Also, CHCA cluster formation is seen with both the solid and

liquid matrices.

Figure 3-10 shows P14R analyzed with both solid and liquid matrices.






80


1000

ACTH 18-39 Solid
Solid

750




U 500[M+H]+




250 [M+CHCA+H]+

[M+2CHCA+H]+


1000

Liquid

750




C 500 [M+H]+




250
[M+CHCA+H]+

[M+2CHCA+H]+

0 A. A. P AI A _
2000 2200 2400 2600 2800 3000
m/z


Figure 3-9. Mass spectra of ACTH fragment 18-39 comparing solid and liquid matrix
preparations. Twenty-five picomoles of analyte was deposited on target, and
each spectrum was the summation of 100 individual spectra.






81


2000

P14R Solid


1500




| 1000 [M+H]
[M+H]+




500




0 .L
2000


[M+H]+ Liquid

1500




C 1000




500

[M+Na]+ [M+DEA+H]+



1100 1300 1500 1700 1900 2100
m/z


Figure 3-10. Mass spectra of P14R comparing solid and liquid matrix preparations.
Twenty-five picomoles of analyte was deposited on target, and each spectrum
was the summation of 100 individual spectra.









For the synthetic peptide, fragmentation is known to occur under mild

conditions.149 Both matrix formulations show y series ions. This ion series is the c-

terminal ion series formed during fragmentation of the peptide, so it contains the c-

terminal and extensions from this residue (i.e., yl4 represents PPPPPPPPPPPPPR).

While different matrix chromophores have shown more or less fragmentation, it appears

that the liquid formulation has fewer fragments present, again indicating a softer

mechanism for desorption and ionization.150

Quantitation

The liquid matrix acts as a homogeneous sampling environment for desorption and

ionization, thus offering opportunity for quantitative analysis. To evaluate the

reproducibility of liquid sampling, intra and inter-sampling precision was determined.

With a liquid matrix sample lasting tens of minutes, ten 1 minute summed spectra could

be compared. Inter-sampling precision was calculated to be -10-13% RSD, with intra-

sampling precision at -5-7% RSD.

Quantitation capabilities, without an internal standard, were examined by

producing calibration curves for angiotensin II and bradykinin fragment 1-7. Figure 3-11

shows the calibration curve obtained for angiotensin II using a liquid matrix. The curve

was obtained by analyzing serial dilutions of a standard peptide mixture, with the volume

of analyte placed on target maintained at 0.5 itL. For Figure 3-11, each point represents a

one minute sum with standard deviations obtained from five analyses. The inset shows a

scaled section of the curve for the femtomole to low picomole range. While the R2

values demonstrate the ability for direct quantitation, the dynamic range for MALDI

becomes apparent when viewing both ranges in Figure 3-11. For this reason, two

analytical functions are presented, one for each range.