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Study of the Conformation of Myoglobin Adsorbed on Nanoparticles Using Hydrogen/deuterium Exchange Mass Spectrometry

Permanent Link: http://ufdc.ufl.edu/UFE0041357/00001

Material Information

Title: Study of the Conformation of Myoglobin Adsorbed on Nanoparticles Using Hydrogen/deuterium Exchange Mass Spectrometry
Physical Description: 1 online resource (94 p.)
Language: english
Creator: Long, Yaoling
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: adsorption, conformation, deuterium, exchange, hydrogen, mass, myoglobin, nanoparticles, protein, spectrometry
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This thesis reports the study of the conformational change of myoglobin adsorbed on nanoparticles using hydrogen/deuterium (H/D) exchange mass spectrometry. The peptide identification and sequence mapping were carried out for myoglobin in solution as well as adsorbed on silver and nickel nanoparticles. H/D exchange of myoglobin in solution was performed and the results were compared with those in literature. Two different enzymes, pepsin and protease XIII, were evaluated as fragmentation agents prior to the mass spectrometric analysis of myoglobin. Compared to pepsin, protease XIII produces peptide fragments of myoglobin in different patterns, as well as more short residues. Hydrogen/deuterium exchange for myoglobin on silver nanoparticles was investigated first on a 14.5 T Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer and then on a Linear Trap Quadrupole (LTQ)-Orbitrap mass spectrometer. When preparing samples for the 14.5 T FTICR, contamination was observed. The sources of contamination were identified using the gel electrophoresis technique. Preliminary H/D exchange mass spectrometry experiments for myoglobin on nanoparticles were performed later using the LTQ-Orbitrap mass spectrometer. The mass increases of peptide residues for myoglobin adsorbed on silver nanoparticles were significantly greater than those observed when myoglobin is in solution. The preliminary results indicate that the method development reported in this thesis is very promising for the investigation of protein conformational changes on nanoparticles.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yaoling Long.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Eyler, John R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041357:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041357/00001

Material Information

Title: Study of the Conformation of Myoglobin Adsorbed on Nanoparticles Using Hydrogen/deuterium Exchange Mass Spectrometry
Physical Description: 1 online resource (94 p.)
Language: english
Creator: Long, Yaoling
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: adsorption, conformation, deuterium, exchange, hydrogen, mass, myoglobin, nanoparticles, protein, spectrometry
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This thesis reports the study of the conformational change of myoglobin adsorbed on nanoparticles using hydrogen/deuterium (H/D) exchange mass spectrometry. The peptide identification and sequence mapping were carried out for myoglobin in solution as well as adsorbed on silver and nickel nanoparticles. H/D exchange of myoglobin in solution was performed and the results were compared with those in literature. Two different enzymes, pepsin and protease XIII, were evaluated as fragmentation agents prior to the mass spectrometric analysis of myoglobin. Compared to pepsin, protease XIII produces peptide fragments of myoglobin in different patterns, as well as more short residues. Hydrogen/deuterium exchange for myoglobin on silver nanoparticles was investigated first on a 14.5 T Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer and then on a Linear Trap Quadrupole (LTQ)-Orbitrap mass spectrometer. When preparing samples for the 14.5 T FTICR, contamination was observed. The sources of contamination were identified using the gel electrophoresis technique. Preliminary H/D exchange mass spectrometry experiments for myoglobin on nanoparticles were performed later using the LTQ-Orbitrap mass spectrometer. The mass increases of peptide residues for myoglobin adsorbed on silver nanoparticles were significantly greater than those observed when myoglobin is in solution. The preliminary results indicate that the method development reported in this thesis is very promising for the investigation of protein conformational changes on nanoparticles.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yaoling Long.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Eyler, John R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041357:00001


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1 STUDY OF THE CONFORMATION OF MYOGLOBIN ADSORBED ON NANOPARTICLES USING HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY By YAOLING LONG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFIL LMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Yaoling Long

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3 To my family

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4 ACKNOWLEDGMENT S I would like to thank my advisor, Dr. John R. Eyler, for his great support and guidance during my graduate study. Dr. Eyler taught me not only how to do scientific research, but also how to be a person that can be helpful to others and to this society. Dr. Eyler is like a great friend. I still remember the first time when he talked to me about this pr oject. I want to thank for all his advices and help in every aspect of my life I would like to thank my co advisor, Dr. David S. Ba rber, for his generosity offering me the opportunity to work on this project. His support, encouragement, help and suggesti ons have been critical in continuing my research. The travel experience to the University of South Florida with him will be good memories to me. I would like to thank my committee member, Dr. David H. Powell, for his great suggestions and help. I have to admit that I should have cherished the time to learn more from him. Appreciation is given to Dr. Benjamin W. Smith for his advice and guidance and Ms. Lori Clark for her patience and support I want to express my gratitude to Dr. Mark Emmett at Florida Sta te University, and Dr. Stan Stevens at the University of South Florida, for their great help offering the time and instrument on sample analysis. Dr. Jeremiah Tipton at Florida State University also provided great help and suggestions. I also want to thank Dr. Nick Polfer for his precious comments and discussions on the project and on my presentation. I would like to thank Dr. Jodie Johnson for his great patience in teaching me the fundamental knowledge on mass spectr ometry I would like to thank Scott Wasd o for the adsorption isotherm experiment, many thanks to Roxanne Werner, John Munson, Ke vin Kioll April Feswick Nick Doperalski in the Center for Human and Environmental

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5 Toxicology, and other friends. Their friendship and help will be my long time treasure. I would like to thank all m y group members also: Michelle, Julia, Jhoana, Sarah, Joanna, Lee, and Cesar for their kind support during my graduate study Enough thanks could not be said to my h usband, my son, and my parents for their moral support gui dance, encouragement, and most importantly, love.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF FIGURES .............................................................................................................. 8 LIST OF ABBREVIATIONS .............................................................................................. 10 ABSTRACT ........................................................................................................................ 13 C H APT ER 1 INTRODUCTION ........................................................................................................ 15 Backg round ................................................................................................................. 15 Motivation .................................................................................................................... 17 Objectives and Approaches ....................................................................................... 18 Outline of the Thesis ................................................................................................... 18 2 LITERATURE REVIEW .............................................................................................. 19 Protein Structures ....................................................................................................... 20 Studies of Protein Conformation ................................................................................ 21 Methodology ......................................................................................................... 21 Circular dichroism .......................................................................................... 21 Fluorescence ................................................................................................. 22 Differential scanning calorimetry ................................................................... 22 Hydrogen/Deuterium exchange .................................................................... 23 H/D exchange mechanisms .......................................................................... 23 Nuclear magnetic resonance spectroscopy ................................................. 26 Mass Spectrometric Methods .............................................................................. 27 General Procedures ...................................................................................... 27 Instrument ...................................................................................................... 28 Study of Protein Conformation on Nanoparticles ...................................................... 30 3 MASS SPECTROMETRIC INVESTIGATION OF MYOGLOBIN IN SOLUTION AND ON NANOPARTICLES ...................................................................................... 35 Sequence Mapping and H/D Exchange of Myoglobin in Solution ............................ 35 Experimental Section ........................................................................................... 35 Materials ........................................................................................................ 35 Instrument ...................................................................................................... 36 Procedure ...................................................................................................... 36 Results and Discussion ........................................................................................ 37 Binding Isotherms of Myoglobin on Nanoparticles .................................................... 41 Experimental Section ........................................................................................... 41

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7 Materials ........................................................................................................ 41 Procedure for generation of protein adsorpt ion isotherms .......................... 42 Results and Discussion ........................................................................................ 42 Pepsin Digestion ......................................................................................................... 44 Experimental Section ........................................................................................... 44 Materials ........................................................................................................ 44 Instrument ...................................................................................................... 44 Analysis of protein digested with pepsin ...................................................... 45 Results and Discussion ........................................................................................ 45 Protease XIII Digestion ............................................................................................... 46 Experimental Section ........................................................................................... 46 Materials ........................................................................................................ 46 Instrument ...................................................................................................... 47 Analysis of protein digested with protease XIII ............................................ 47 Results and Discussion ........................................................................................ 47 Summary ..................................................................................................................... 48 4 H/D EXCHANGE MASS SPECTROMETRY OF MYOGLOBIN ON SILVER NANOPARTICLES ..................................................................................................... 69 Analysis for Sample Contamination Source .............................................................. 69 Sample Preparation for HDEX -MS ...................................................................... 69 Materials ........................................................................................................ 69 Instrument ...................................................................................................... 70 H/D exchange mass spectrometry of protein i n solution ............................. 70 H/D exchange mass spectrometry of protein on Ag nanoparticles ............. 70 Gel electrophoresis analysis for sample contamination ............................... 71 Results and Discussion ........................................................................................ 71 HDEX -MS of Myoglobin using LTQ -Orbitrap ............................................................. 74 Experimental Section ........................................................................................... 74 Materials ........................................................................................................ 74 Instrument ...................................................................................................... 74 Analysis of protein digested with protease XIII ............................................ 74 H/D Exchange mass spectrometry of protein on Ag nanoparticles ............. 75 Results and Discussion ........................................................................................ 76 5 CONCLUSIONS AND FUTURE WORK .................................................................... 89 Conclusions ................................................................................................................ 89 Future Work ................................................................................................................ 90 LIST OF REFERENCES ................................................................................................... 91 BIOGRAPHICAL SKETCH ................................................................................................ 94

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8 LIST OF FIGURES Figure page 2 -1 Far UV CD spectra of pr oteins with different structures. ...................................... 32 2 -2 Protein H/D exchange mechanisms via local unfolding or global unfolding ....... 33 2 -3 Intrinsic chemical H/D exchange rate as a function of pH .................................... 33 2 -4 15N -HSQC NMR spectra of DnaK before and after H/D exchange ..................... 34 2 -5 Experimen tal procedure of protein H/D exchange using mass spectrometry ...... 34 3 -1 Mass spectra of myoglobin in solution obtained with ESI -Q -TOF ........................ 50 3 -2 Expansion of 46 4 to 476 m/z region of Figure 31 ................................................ 51 3 -3 ................................................... 52 3 -4 ................... 53 3 -5 Mass spectra of myoglobin in solution obtained after different digestion times ... 54 3 -6 Mass spectra of myoglobin in solution obtained after different digestion times ... 55 3 -7 Comparison of peptide coverage after different digestion times .......................... 56 3 -8 Comparison of peptide coverage after different digestion times .......................... 57 3 -9 Mass spectra showing mass shifts during H/D exchange .................................... 58 3 -10 Deuterium uptake of various residues in myoglobin in solution ........................... 59 3 -11 Comparison of mass increase for 6 residues ........................................................ 60 3 -12 Comparison of the change of peak width in H/D exchange.................................. 61 3 -13 Mass increase simulation for residue 1-29 and 3069 .......................................... 62 3 -14 Binding isotherms of myoglobin on nanoparticles ................................................. 63 3 -15 Mass spectra of myoglobin digest in solution and on nanoparticles .................... 64 3 -16 Sequence mapping of myoglobin in solution and on nanoparticles, after pepsin digestion ..................................................................................................... 65 3 -17 Mass spectrum of intact apomyoglobin ................................................................. 66

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9 3 -18 Sequence mapping of myoglobin in solution and on silver nanoparticles digested by protease XIII ....................................................................................... 67 3 -19 Common fragments of myoglobin in solution and on nanoparticles digested by protease XIII ...................................................................................................... 68 4 -1 SDS-PAGE of fre shly made myoglobin digests and old samples ........................ 78 4 -2 SDS-PAGE of freshly made myoglobin digests .................................................... 79 4 -3 SDS-PAGE of freshly made myog lobin digests .................................................... 80 4 -4 Mass spectra of myoglobin digests and blank following filtration with 200 nm nylon filter ............................................................................................................... 81 4 -5 Mass spectru m of contaminants from 200 nm nylon filter .................................... 82 4 -6 Mass spectrum of myoglobin digests following filtration with 200 nm Supor membrane............................................................................................................... 83 4 -7 Zoomedin mass spectra of myoglobin digests following filtration with 200 nm Supor membrane .................................................................................................... 84 4 -8 SDS-PAGE of myoglobin digested with protease XIII after 200 nm Supor membrane filtration ................................................................................................. 85 4 -9 Peptide coverage of myoglobin digested with protease XIII ................................. 86 4 -10 H/D exchange mass spectra of myoglobin in solution an d on silver nanoparticles .......................................................................................................... 87 4 -11 Deuterium uptake of myoglobin in solution and on silver nanoparticles .............. 88

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10 LIST OF ABBREVIATION S A Arrhe nius coefficient Ab am ount of protein bound to nanoparticles Amax maximum amount of protein bound to nanoparticles B magnetic field strength BET Brunauer, Emmett and Teller Bis bisacrylamide c concentration CD circular dichroism COSY correlation spectroscopy DC direct circuit D SC differential scanning calorimetry Ea reaction a ctivation energy ESI electrospray ionization FDA Food and Drug Administration FTICR Fourier Transform Ion Cyclotron Resonance HDEX H ydrogen /D euterium exchange HEPES N -2 -hydroxyethylpiperazine -N 2 ethanesul fonic acid HMC heated metal capillary HPLC High performance liquid chromatography HSQC heteronuclear single quantum coherence ICR ion cyclotron resonance k chemical reaction rate constant k Langmuir adsorption constant k1 unfolding rate constant

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11 k1 foldin g rate constant k2 intrinsic chemical exchange rate constant kex hydrogen/ deuterium exchange rate constant kobs observed rate constant LTQ Linear Trap Quadrupole m1 exchange hydrogens in side chain and terminal of peptides m2 exchangeable hydrogens on amid e bonds in peptides MS mass spectrometry NHMFL National High Magnetic Field Laboratory NMR nuclear magnetic resonance NOESY nuclear o verhauser effect spectroscopy PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline QIT quadrupole ion trap Q -TOF quadrupole-time of flight R universal gas constant RF radio frequency SDS Sodium dodecyl sulfate T Tesla t hydrogen/ deuterium exchange time TOF time of flight Tris tris(hydroxymethyl)aminomethane Trp Tryptophan UV Ultra Violet ellipticity (in degrees)

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12 percentage of deuterium atoms for all hydrogen isotope atoms M mass increase after hydrogen/deuterium exchange

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13 Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science STUDY OF THE CONFORMATION OF MYOGLOBIN ADSORBED ON NANOPARTICLES USING HYDROGE N/DEUTERIUM EXCHANGE MASS SPECTROMETRY By Yaoling Long December 2009 Chair: John R. Eyler Major: Chemistry This thesis reports the st udy of the conformational c hange of myoglobin adsorbed on nanoparticles using hydrogen/deuterium (H/D ) exchange mass spectrometry. The peptide identification and sequence mapping we re carried out for myoglobin in solution as well as adsorbed on silver and nickel nan oparticles. H/D exchange of myoglobin in solution was performed and the results were compared with thos e in literature. Two different enzymes, pepsin and protease XIII, were evaluated as fragmentation agents prior to the mass spec trometric analysis of myoglo bin. Compared to pepsin, protease XIII produces peptide fr agments of myoglobin in differ ent patterns, as well as more short residues. Hydrogen/deuterium exchange for myoglobin on silver nanoparticles was investigated first on a 14.5 T Fourier Trans form Ion Cyclotron Resonance (FTICR) mass spectrometer and then on a Linear Tr ap Quadrupole (LTQ)-Orbitrap mass spectrometer. When preparing samples fo r the 14.5 T FTICR, contamination was observed. The sources of contamination we re identified using the gel electrophoresis technique. Preliminary H/D ex change mass spectrometry experiments for myoglobin on

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14 nanoparticles were performed later using t he LTQ-Orbitrap mass spectrometer. The mass increases of peptide residues for myoglobin adsorbed on silver nanoparticles were significantly greater than those observed when myoglob in is in solution. The preliminary results indicate that the method developm ent reported in this thesis is very promising for the investigation of pr otein conformational changes on nanoparticles.

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15 CHAPTER 1 INTRODUCTION Background Nanoparticles, particles 100 nm or less in size in at least one dimension, such as metals (gold, silver), metal oxides (TiO2, SiO2), inorganic materials (carbon nanotubes, quantum dots), and polymeric materials, have re ceived considerable attention recently in both diagnostics and therapeut ics, primarily due to their potential benefits in the specificity of ta rgeted drug delivery1, 2. Nanoparticles have also found wide application in many other fields, including cosmetics, el ectronics, and the automot ive industry. Under those manufacturing circumstances, it is not surprising that nanoparticles may enter into the bodies of workers who are in close cont act with them, by inhalation, dermal contact or ingestion. For both of these reasons, the toxicological impacts of nanoparticles on human health are of concern3. However, currently there is no clear understanding of the mechanisms of nanoparticle behavior in the human body. Nanoparticles primary action in human body may be initiated by the adsorption of certain serum proteins on the particles surfaces4, 5. As a general rule, when exposed to proteinaceous environments, such as pl asma, nanoparticles are coated with proteins immediately. Protein adsorption on nanoparticles surfaces may result in surface exposure of residues (cryptic epitopes) t hat are normally hidden in the protein core6. The unfolding of cryptic epitopes could trigge r inappropriate cellular processes, but the protein structure after exposure of these residues is st ill similar enough to that of the native protein so that the ce lls can not recognize the difference. Even after desorption from the nanoparticle surface, some of these conformational changes are irreversible7, 8. For either of the above cases, a clear understanding of the interactions between

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16 nanomaterials and proteins in physiological syst ems is critical for the development of new and more appropriate nanomaterials and fo r evaluation of their toxicity. In particular, a complete understanding of these kinds of interactions may help prevent adverse response in the immune system6, 9 when nanomaterials are used in therapy. Various methods have been used to study th e conformational changes of proteins adsorbed on nanoparticles. These methods in clude molecular light fluorescence, differential scanning calorimetry (D SC), and circular dichroism (CD)10, 11. Although some of these methods are very sensitive to t he protein conformational changes, they have their disadvantages, such as inability to detect 3-dimensional structural changes, susceptibility to interferences, and problems in monitoring residue-specific information. The hydrogen/deuterium exchange technique has been widely used in exploring protein conformation for the past 30 years. Usually H/D exchange is combined with nuclear magnetic resonance (NMR) spectroscopy12, 13. By observing the chemical shifts of specific hydrogens, information at many sites of the prot ein can be obtained. However, NMR usually requires concentrations as high as the mM level, which are often not feasible for many protei ns due to aggregation at these concentrations. It is also difficult to use NMR to study protei ns with very high molecular weights14. Recently, mass spectrometry (MS) co mbined with H/D exchange (HDEX) has been used to explore protein conformation. The benefits of using mass spectrometry include: (1) low detection limits; (2) the capab ility to analyze very large proteins; and (3) the possibility of obtaining local charge information for peptides when using the electrospray ionization (ESI) technique. Usua lly, the resolution is at the level of 5-10 residues, making HDEX-MS a medium resolu tion technique. However, the resolution

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17 may be improved by using various proteases to produce different fragments for the same protein. Motivation Buijs et al.15 used H/D exchange FTICR-MS to study the conformation and dynamics of myoglobin adsorbed on silica nanoparticles. Their results showed that HDEX-MS can be used to detect loca l conformational changes and dynamics associated with protein adsorpt ion on nanoparticles. Many types of materials can be used for nanoparticle synthesis, including polymers, metals, and metal oxides6. Protein adsorption behavior may be affected by a va riety of nanoparticle properties, including shape, size, surface charge and surface composition6, 16. One goal of this research was to investigate the conformational changes of proteins, specifically myoglobin, adsorbed on various nanoparticles. One issue identified in Buijs study was t he sizes of the peptide fragments, which were too large to achieve an adequate residue resolution. Buijs et al. used pepsin to partially digest the protein, but it was not possible to obtain small fragments under their experimental conditions, such as limi ted digestion time. Cravello et al.17 found that a different enzyme, protease type XIII, w hen combined with other enzymes, can give more detailed protein structural informa tion as well as better peptide coverage. A significant benefit is that pr otease type XIII is able to hydrolyze proteins to produce more fragments than pepsin or trypsin17. In addition, protease type XIII exhibits much less self-digestion than pepsin18. Another objective of this re search was to use protease type XIII to study the conformational chan ges of myoglobin on nanoparticles by using HDEX-MS.

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18 Objectives and Approaches The overall objective of this research was to investigate the conformational changes of myoglobin adsorbed on nanoparticles using HDEX-MS. There were two main objectives. The first objective was to investigate H/ D exchange of myoglobin in solution. After repeating the work referenced in literature, bindi ng isotherms of myoglobin on various nanoparticles were obtained. Silver and nicke l nanoparticles were selected as the adsorption substrates. Mass spectrometry was performed for myoglobin in solution and on nanoparticles after protease digestion using protease type XIII and pepsin. The second objective was to investi gate H/D exchange of myoglobin adsorbed on nanoparticles using mass spectrometric methods. Samples were prepared and subjected to mass spectrometric analysis. Reasons for possible contamination were also explored. Outline of the Thesis This thesis consists of 5 chapters. This chapter has introduced background, motivation, and the objectives and approache s of the research. The second chapter reviews the literature related to the current research. The third chapter presents mass spectrometric data on myoglobin in solution and on nanoparticles be fore H/D exchange, as well as H/D exchange results in solution. The fourth chapter discusses the source of contamination in the samples and present s H/D exchange data fo r myoglobin adsorbed on silver nanoparticles. The fifth chapter is the conclusion, including suggestions for future work.

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19 CHAPTER 2 LITERATURE REVIEW Nanoparticles encompass particles with si zes ranging from 1 nanometer up to a hundred nanometers. In general, a decrease in particle size results in changes to physicochemical properties, for example, changes in morphology, electronic and atomic structure, phase transformation1. Due to the unique properti es of nanoparticles, they have been used extensively in various applic ations, including medical diagnosis and therapy development2, 9, 19, 20. Nanotechnology has been listed in the FDA s Critical Path Opportunities Report and List, which was created to provide a pl atform for collaborative work on critical scientific issues in medical product development and patient care21. Specifically, nanoparticles can act as carriers or deliverer s for therapeutic drugs to reach certain targeted organs (cells) in the human body. Various types of nanoparticles have been investigated as drug carriers in treating tumors19, such as ovarian cancer22 and liver cancer9. Nanoparticles have already found wide application in many other fields, including the cosmetics, electronics, and automotive industries. Humans may be exposed to nanoparticles through inhalation, dermal cont act, or ingestion. Some of these nanomaterials have been shown to be toxic23. However, the impacts on human health by such exposure are still not fully understood3. The properties of nanoparticles, such as size, shape, composition, surface area, surface charge etc, may be of importance in understanding possible toxic effects. It is generally agreed that proteins will adsorb onto the surfaces of nanoparticles as soon as the particles enter biological fluids4. Upon adsorption onto the nanoparticle

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20 surface, protein conformational changes may occur10, 24, 25. Different from those of flat surfaces, the greater curvature of t he smaller nanoparticles may cause greater disruption to protein conformation6. Some of these changes may be irreversible7, 8, resulting in alterations in the normal function of proteins. Research focusing on conformational changes in protein adsor bed on nanoparticles is necessary for the understanding of potential toxicological impacts of nanoparticles within the human body. Protein Structures Protein structure can be classified into 4 levels. The primary structure refers to the amino acid sequence of the pept ide chains of a protein molecule. It is unique to a specific protein. Primary stru cture sometimes is called coval ent structure, since all of the covalent bonds in protein are included in primary structure, except for disulfide bonds. The secondary structure usually refers to the regular local g eometry of the main peptide chains in the protein. Alpha-helix and betasheet are common secondary structures. The formation of the secondary st ructure is mainly controlled by hydrogen bonding within the peptide backbone. As com pared with the secondar y structure, the tertiary structure pertains to the global, 3dimensional geometry showing how a peptide chain folds in such a way that hydrophobic am ino acid residues are hidden or buried within the structure, whereas hydrophilic residues are exposed on the outer surface. The formation of tertiary structure is cont rolled by non-covalent interactions, including hydrogen bonding, van der Waals forces, and ionic interactions. Beside the primary structure of amino acid s equences, the tertiary struct ure usually determines the biological activity of a protei n. Conformational study of prot eins usually refers to the study of secondary and tertiary structures The quaternary structure is the stable association of 2 or more polypeptide c hains, where individual chains are called

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21 subunits. Quaternary structure is normally stabilized by non-covalent interactions, including hydrogen bonding, van der W aals forces, and ionic bonding, between subunits. Not all proteins have quaternary structure. Studies of Protei n Conformation Methodology Various methods have been employed to study protein conformations. However, none of the methods is perfe ct for all purposes. Every method has its advantages and disadvantages. Circular dichroism Circular dichroism (CD) spectroscopy is one of the most commonly used methods for studying protein conformation. Plane-po larized light can be considered as the resultant of 2 circularly polarized component s rotating in opposite directions. These 2 components can be absorbed to different extents if the molecule is chiral, which is the case for protein molecules. Under such condi tions, the transmitted light is said to have elliptical polarization represented by the ellipticity ( ) in degrees. The differential absorption of the polarized in cident light can be used as a probe for showing different folding or unfolding patterns of protein molecules. Figure 2-126 shows far-UV CD spectra for various types of protein secondary struct ure. Circular dichroism can give information about secondary structure (far-UV CD), such as alpha-helix and beta-sheet formation, as well as a fingerprint of the tertiary st ructure (near-UV CD). However, when studying proteins adsorbed on nanoparticl es, data must be interpret ed with caution, because CD is very sensitive to interferences from solid particles, which can scatter the incident light. Experience in operating the instru ment is also necessary to ob tain satisfactory results.

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22 Fluorescence Fluorescence is another popular spectroscopi c technique for the study of protein conformations5, 11, 27. Tryptophan (Trp), an ar omatic amino acid residue that occurs in most proteins, fluoresces when excited with the appropriate wavelength of UV light. The fluorescence of Trp is highly sensitive to the surrounding environment. For a protein in its native state, Trp is often hidden in the hydrophobic core When the protein unfolds, Trp may be exposed to a more hydrophilic environment, resulting in a red shift (shift to longer wavelengths) of the fluorescence due to the increased dielectr ic constant of the surrounding medium. Based on this principl e, theoretically Trp fluorescence can be used to monitor the conforma tional changes of proteins, even at atomic resolution27. However, in practice, many factors can affe ct fluorescence signatures of proteins, and it is usually difficult to obtain a det ailed structural interpretation. Differential scanning calorimetry Spectroscopic methods, such as CD, fluore scence, or infrared, often suffer from interferences due to light scattering. A non-s pectroscopic method, differential scanning calorimetry (DSC) is often used as a complementary method5, 28-31. The principle of DSC is based on the changes of heat capacity resulting from protein conformational changes. In practice, DSC measures heat c apacity of the sample as a function of temperature. Therefore, DSC can directly obtain thermodynamic data about protein conformation and stability. However, comp ared to the spectroscopic methods, DSC gives a general overall measure of the proteins energetics. It cannot give site specific information related to location and t he nature of structural changes.

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23 Hydrogen/Deuterium exchange The technique of amide hydrogen/deuter ium (H/D) exchange has been a powerful and popular tool in studying protein struct ure and dynamics since the early work by Berger and Linderstrom (1957). Labile hydrogen atoms, which include amide hydrogens, can undergo isotopi c exchange with a deuterated solvent (usually deuterated water). This process can be monitored using various instrumental techniques including nuclear magnetic re sonance (NMR), and more recently, mass spectrometry (MS). In protein molecules, several types of hydrogens can possibly undergo exchange: polar side-chai n hydrogens attached to heteroatoms (N, O, and S), hydrogens at both the Nand Ctermi ni, and peptide backbone amide hydrogens. However, the exchange rates vary signi ficantly. Side-chain and terminal hydrogens usually exchange at rates much faster t han those of amide hydrogens at normal physiological conditions32, 33 and its often beyond the capabi lity of current detection techniques to detect these side-chain and terminal hydrogens. Thus, conventionally H/D exchange in proteins refers specifically to amide H/D exchange. Fortunately, protein conformational stability and secondary structur e have significant effects on the amide hydrogens which are involved in hydrogen bonds as part of protein secondary structure and protein folding. The exchange rate fo r a specific amide hydrogen can provide detailed structural and stability in formation on that specific peptide. H/D exchange mechanisms As early as the 1950s, H/D exchange wa s used to study protein structure and conformational stability34, 35. H/D exchange can result from small local structural fluctuations or solvent penetration, revers ible local unfolding or global unfolding reactions. Figure 2-2 gives pictorial repr esentations for two proposed mechanisms for

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24 H/D exchange. In t he dual-pathway model32, protein molecules undergo H/D exchange directly from the folded state. Amide hydrogens at or near the surface of the protein are assumed to exchange by this pathway via small local transient openings. These hydrogens usually do not invo lve any hydrogen bonding or protection from solvents. H/D exchange mediated by reversible local fluctuations or unfolding and /or global unfolding is more often described by a second pathway32, 33. In this mechanism, the folded protein molecule undergoes unfolding initially, either loca lly (as in (A)) or globally (as in (B)), followed by H/D exchange wit h the surrounding solvent, and completes the exchange process after re-folding. The se cond pathway (so-called Linderstrom-Lang model) can be further classified into 2 types: EX1 and EX2. Generally, in either EX1 or EX2, the overall observed rate constant for H/D exchange can be written as12: (2-1) Where and are defined in Figure 2-2. EX1 mechanism: The EX1 mechanism predominates when the chemical intrinsic exchange rate constant ( ) is much faster than the refolding rate constant ( ). The observed exchange rate constant ( ) will be directly proportional to and limited by the unfolding rate constant ( ): (2-2) This occurs when the inter-conversion of t he folded and unfolded conformations is very slow, such as in the condition when a denaturant is added, or when the reaction conditions (pH, temper ature, etc) change. The intrinsic chemical rate of H/ D exchange on amide hy drogens is greatly affected by pH, temperature, and soluti on composition. The amide H/D exchange

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25 reaction can be catalyzed by both acid and base. The exchange has the slowest rate around pH 2-3 (Figure 2-3). According to t he Arrhenius equation, the rate constant, is also affected by temperature T Based on the activation energies ( Ea) found in Bais study36, the exchange rate decreases 3-fold with every 10K decrease in temperature. The exchange rate can be r educed by up to 5 orders of magnitude by changing the pH from 7 at 25 to 2.5 at 0 This large difference in the exchange rate at different pHs and temperat ures is the basis of the experimental procedure in H/D exchange study using mass spectrometry which will be introduced later. EX2 mechanism: When the rate constant for refo lding is much greater than the intrinsic chemical exchange rate constant, the observed rate can be simplified to: (2-3) Under this condition (called the EX2 mec hanism), the observed exchange rate depends on the equilibrium of the locally folding and unfolding states. If all the amide hydrogens have the same stability, i.e., the protein structure is unifo rmly stable along the peptide chain, H/D exchange would not provide much information about the protein stability, since there would be no difference in terms of the H/D exchange ra tes. Fortunately, protein local stability varies significantly and there are always some regions that are less stable than other regions. The local stabi lity can also be changed by changing conditions. The less stable regions will have fa ster exchange rates. It is possible to obtain structural and conformational st ability information using the H/D exchange technique. The EX2 mechanism is mostly observed under usual physiological conditions. Switching from EX2 to EX1 may occur when reaction conditions, such as pH, change significantly, to fa vor the intrinsic chemical reaction rate of the exchange.

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26 Nuclear magnetic resonance spectroscopy Combined with H/D exchange, NMR has been widely used in protein conformational studies37. The basic principle is t hat different hydrogen atoms (1H, spin of ) have different chemical shifts, while deuterium atoms (2H, spin of 1) are not detected at the frequenc ies used to observe 1H. Before performing H/D exchange, the chemical shift assignments of specific am ide hydrogens are determined according to prescribed strategies38. Then D2O is added, and the hydrogens are allowed to exchange. By analyzing the disappearance and the change in peak intensity or area of amide hydrogen resonances in the 1H NMR after H/D exchange, information about specific amide hydrogens stability and structure can be obtained. NMR can be used to study thermodynamic properties39, folding intermediates40, and conformational dynamics12 in proteins. With the development of NMR technology, several NMR methods can be used to monitor amide hydrogen exchange in proteins12. One-dimensional 1H NMR is reasonably easy to understand and perform. However, multi-dimensional NMR techniques, such as correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum coherence (HSQC), are more useful in determining the properties of site-specific amide hydrogens. The main benefit of multi-dimens ional NMR spectroscopy is t he correlation of hydrogens with other atoms in close prox imity. For example, in 1H-1H COSY, amide hydrogens can be correlated with hydrogens on their alpha-carbons. In 15N-HSQC, amide hydrogens are correlated with attached nitrogen atoms in 15N-labeled proteins. Spectral assignment can be greatly simplified by using multi-di mensional NMR, which shows how atoms are correlated through bonds or through space. Figure 2-441 shows a comparison of 15N-

PAGE 27

27 HSQC spectra of DnaK (a protein with 638 amino acids and molecular weight of 70 kDa) in D2O at time 0 and 24 hours. After 24 hour s of exchange, some signals have disappeared, indicating that the hy drogens bonded to those nitrogens have been exchanged. One disadvantage of NMR is that it usually requires high concentrations (normally at the mM level) of protein for the analysis, which is often impractical for many proteins with high agglomeration tendency. Another disadvantage is that NMR does not provide high resolution and accuracy for proteins la rger than 30 kDa, due to resonance overlap and peak broadening. Mass Spectrometric Methods Since the 1990s, mass spectrometric me thods used in conjunction with H/D exchange (HDEX-MS) have gained more and more popularity in protein conformational studies. Compared to the traditional NMR me thods, MS methods are not site-specific, i.e., they cannot study the H/D exchange on individual amino acid residues. However, MS methods provide much better sensit ivity and require much lower protein concentration ( mol to submol). MS methods can also analyze large proteins with molecular weight s greater than 106 Da. General Procedures The underlying principle of HDEX-MS is t he increase in mass that occurs when 2H exchanges for 1H. By analyzing the amount of deuterium uptake in protein molecules or peptide fragments, information on protein conformational stability and exchange dynamics can be obtained. In experimental terms, a general procedure for protein HDEX-MS is shown in Figure 25 and can be listed as follows: A concentrated protein solution is made with protic water.

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28 A portion of concentrated protein soluti on is diluted in deuterated water. The dilution ratio is usually at least 1:10 H2O:D2O. The diluted solution is incubated for certain amount of ti me depending on the desired extent of H/D exchange. An aliquot of the solution is removed and co ld acid is added to decrease the pH to ~2 2.5, with the temperature kept at 0 to quench the exchange reaction. An enzymatic protease solution is added to the quenched solution to digest the protein into peptide fragments. The digested solution is injected into a reversed phase HPLC column and the peptides are separated. The peptides are analyzed by a mass spectrometer. In HDEX-MS, electrospray ionization (ESI)42 is the usual method of introducing charged samples into the mass s pectrometer due to the ability to obtain charged intact peptides, thus greatly simplifying the spectr um and the identification process. In this sense, ESI is considered a soft ionization met hod. Another feature of ESI is that several H+1s can be added during the ESI pr ocess, which means that the same molecule can have multiple peaks at different m/z values. By analyzing the charge-state distribution, the presence of a certain analyte can be conf irmed. Additionally, el ectrospray ionizes molecules with almost no mass limitation. Even with very large molecules, the ability to produce multiply-charged ions greatly reduces the m/z value to a range which can be handled by mass analyzers. Instrument The most common mass analyzers used in protein conformation include time-offlight (TOF), Fourier transform ion cyclot ron resonance (FTICR), and most recently, Orbitrap mass spectrometers.

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29 The principle of TOF-MS is simple. I ons with different mass/charge ratios are accelerated to the same kinetic energy by a certain potential. These ions are then introduced into a field-free t ube, along which the ions dri ft toward the detector at different velocities depending on their masses. The travel time is proportional to the square root of the m/z value. Measuring the ion signal intensity as a function of time will produce the mass spectrum. In an FTICR spectrometer, ions are trapped in a limited volume (cell) by applying electrostatic and magnetic fields. The Lorentz forc e induces the ions to move in circular cyclotron paths in the electrostatic field and the magnetic field. The frequency of this cyclotron motion is uniquely depe ndent on the magnetic field str ength, B, and the m/z of the ion. The cyclotron motion in the cell produc es an image current which oscillates at a frequency identical to that of the ions. Measurement of the image current can be used to deduce the mass spectrum after Fourier transformation of t he raw data acquired in the time domain43. An Orbitrap is also an ion trap, similar to a quadrupole ion tr ap (QIT) or ICR ion trap, with neither a magnetic field nor a radio frequency (RF) oscillating electric field applied to the ions44, 45. Conceptually, an Orbitrap consists of a spindle-like central electrode and a barrel-like outer electrode. A DC voltage is applied between the two electrodes. Ions that are injected into the trap will have frequencies along 3 directions under the electric field: frequency of ion ro tation around the central electrode, frequency of radial oscillation and fr equency of axial oscillation along the central electrode. Among the 3 frequencies, only the frequency of axia l oscillation is independent of an ions properties, such as kinetic energy, injecti on angle, etc. This frequency is inversely

PAGE 30

30 proportional to the square root of m/z value of the ions and is used for mass analysis. The axial oscillation induces an image current on the outer electrode. After Fourier transformation, the measurement of this image current can be used to obtain the mass spectrum. Among these mass analysis techniques, FTICR has the best mass accuracy and resolution, but its instrumentation is al so the most expensive and requires regular maintenance. The Orbitrap has similar mass accuracy and resolution to those of FTICR instruments, but it does not need a supe r-conducting magnet and is more compact and less costly. Study of Protein Confo rmation on Nanoparticles As discussed previously, adsorption of proteins on the nanoparticle surface leads to conformational changes, which possibly wil l cause a significant impact on protein activity, as well as the proteins intera ction with other proteins (protein-protein interaction). Partial unfolding of protein may provoke adverse effects, such as over expression of inflammatory fa ctors (for example, cytokines46) or reactive oxygen species47. Due to this, it is of critical impor tance to investigate the conformational changes of proteins adsorbed on nanoparticl es. There have been a fair number of publications5, 25, 29, 31 studying the conformational st ability and dynamics of proteins adsorbed on nanoparticles using methods such as CD, fluorescence, and NMR. These methods have proven useful, but with the limitations described above. Although mass spectrometric methods can overcome those limitations, such studies have been rare. Buijs et at.15 investigated the conformational stab ility of various local regions of myoglobin adsorbed on silica nanoparticles using H/D exchange coupled with FTICR MS. Myoglobin is a single-chain protein with 153 amino acid residues. It usually has an

PAGE 31

31 iron-containing heme group in the center, around which apomyoglobin folds. Myoglobin is a common model protein which has been extensively studied for many years. Much information has been obtained about myoglobin that could be very helpful for this research. Two peptide fragment s (residue 30-69 and residue 70106), which are located in the middle of the protei n chain and close the heme gr oup, did not show stability changes after adsorption onto a nanoparticle15. The two terminal fragments (residue 129 and residue 107-137), however, were destabilized upon adsorption. Two major issues deserve further investigati on. First, the residue resolution in the Buijs et al. study was not high. The peptide fragments were too large to obtain any further local specific info rmation. It would be desirable to produce smaller peptide fragments during digestion. Second, different s ubstrates and proteins should be used to extend the scope of the research. This thesis attempts to address these two issues. Two enzymatic proteases, pepsin and type XIII, were employed durin g the digestion and compared in terms of their fragmentation efficiencies and styles Several metal-based nanoparticles were explored as the adsorption substrate. Silver nanoparticles were selected for most of the study.

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32 Figure 2-1. Far UV CD spectra of proteins wit h different structures. Solid line, a-helix; long dashed line, anti-parallel -sheet; dotted line, type I -turn; cross dashed line, extended 31-helix or poly (Pro) II helix ; short dashed line, irregular structure. The figure is used wit h permission of Elsevier B. V. -helix Anti-parallel -sheet

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33 Figure 2-2. Protein H/D exchange mechanisms via local unfolding (A) or global unfolding (B) Figure 2-3. Intrinsic chemical H/D exchange rate as a function of pH k2D2O H D k2D2O k-1k1 H k1k-1 H D D H k1k-1 k-1k1 D (A) (B)

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34 Figure 2-4. 15N-HSQC NMR spectra of DnaK before and after H/D exchange. (A) Before H/D exchange; (B) After H/ D exchange. The figure is used with permission of John Wiley & Sons. Figure 2-5. Experimental pr ocedure of protein H/D exch ange using mass spectrometry H H H H H H H H D D D D D H H D D D D D D D H H D2O buffer Quench 0 pH 2.5 Protease Digestion Relative Intensity No deuteration After deuteration m/z Peptides SeparationHPLC MS Calculate Deuterium Uptake m D H D D H D D D A B

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35 CHAPTER 3 MASS SPECTROMETRIC INVESTIGATION OF MYOGLOBIN IN SOLUTION AND ON NANOPARTICLES As stated in the previous chapter, t he conformation and exchange kinetics of myoglobin adsorbed on silica nanoparticles have been studied using FTICR MS. Pepsin was used as the protease, and peptide fragment s including amino acid residues 1-29, 30-69, 70-106, and 107-137 were analyzed. In this chapter, mass spectrometric data about myoglobin in solution as well as adsorbed on nanoparticles are presented. Firs t, experiments were carried out with the objective of comparing results to literature values15. Second, binding isotherms of protein adsorbed on various nanoparticles were obtained. The objective was to find an appropriate nanoparticle material for the HD EX-MS experiment. After analyzing the binding isotherms, pepsin and protease type XIII were used as the proteases. Results of peptide sequence mapping are pr esented in this chapter. Sequence Mapping and H/D Exchange of Myoglobin in Solution This section reports the re sults of experiments in wh ich myoglobin in solution was analyzed using ESI-Q-TOF mass spectrometry Protein peptide sequence identification results showed similarity to literature values15, and reproducible coverage was also obtained. Although the results were similar to those in the reference, the peptide sequence was a little different, because a myogl obin from a different species was used. Experimental Section Materials Myoglobin from equine skeletal muscle was purchased from Sigma Aldrich. Porcine gastric pepsin A was purchased from Biochemical Corporation. 5 mM HEPES

PAGE 36

36 (N-2-hydroxyethylpiperazine-N-2-ethanesulf onic acid) solution (Fisher Biotech) was used as the buffer, pH 6.5. Heavy water was 99.9% D2O (Sigma Aldrich). Instrument The ESI-Q-TOF mass spectrometer wa s a QStar XL (Applied Biosystems) equipped with an ion spray source and using N2 as the nebulizing and desolvation gas. The applied cone voltage range was from 2 000 to 2500 V, and no source heater was needed. The scanned range was from m/z 400 to 1500, and samples were scanned for 1 minute (60 total scans) or 2 minutes (120 total scans). Procedure Myoglobin in solution: First, 100 L of 50 M myoglobin in 5 mM HEPES was digested with 650 L of 6 M pepsin in 6% acetic acid for 2 minutes at 0 Then 650 L of ice-cold methanol was added, and the sample was introduced into the mass spectrometer. The final myoglobin concentration was 3.6 M. For comparison, a more concentrated sample was prepared by digesting 100 L of 300 M myoglobin in 5 mM HEPES with 650 L of 36 M pepsin in 6% acetic acid for 2 minutes at 0 then 650 L of ice-cold methanol was added and the sample was introduced into the mass spectrometer. The final myoglobin concentration was 21 M. H/D exchange of myoglobin in solution: A 10 L aliquot of 1mM myoglobin in 100mM HEPES was mixed with 190 L of D2O. The mixture was incubated for different lengths of time to allow for H/D exchange. A 10 L aliquot of this solution was mixed with 65 L of 6 mM pepsin in 6% acetic acid at 0 for 2 minutes, followed by the addition of 65 L of ice-cold methanol. The mixt ure was infused into the mass spectrometer immediately.

PAGE 37

37 Peptide identification. The MS-fit program with the SwissProt (06/10/2008) database was used to identify the digested p eptides of myoglobin with porcine gastric pepsin as the protease. Accession number of myoglobin was P68082. The maximum of the missed cleavages was set up as 15. Monoisotopic masses obtained from the spectra were input into the program to compare with the calculated data with 20 ppm mass error tolerance. ( http://prospector.ucsf.edu/prospector/cgibin/msform.cgi?form=msfitstandard last accessed on October 4, 2009). Results and Discussion The mass spectrometric reproducibility under different protein concentrations and digestion times was investigated. Full scan spectra and sequence mapping and coverage were compared. The complete spectra of the digested my oglobin at two concentrations are shown in Figure 3-1. There is no significant difference between the 2 spectra in terms of the position and the relative int ensities of the major peaks. The m/z range from 464 to 476 Da of Figure 3-1 is shown in Figure 3-2. It can be clearly seen that the peak clusters have sim ilar isotopic distribution patterns, and for the most part, the relative intensities am ong peaks are similar. The 70-103 residue has higher relative intensity in the 21 M solution than in the 3 M solution. This may be due to the different rates of digestion among di fferent parts of the chain. In general, however, the concentration does not signifi cantly affect the final spectra. Figure 3-3 shows peptide residue peaks in the digested myoglobin: residues 1-29, 30-69, 70-106, 107-137, and 138-153. These residues cover the length of the molecule, indicating that the coverage is 100% (a better view of the coverage is shown later in the sequence mapping part) from this spectrum. Some of these residues (1-29, 30-69 and

PAGE 38

38 70-106) were analyzed using HDEX-MS and resu lts were compared with the results of Buijs15. For some residues, for example, resi due 70-106, smaller fragments (residue 87106 and 70-86) can be used to study the stru cture of peptide in higher resolution. Usually peptide sequence m apping is used to show the percent peptide coverage and the degree of amino acid resolution provided by the enzyme and the analytical method. With higher coverage, a better underst anding of the proteins global structure can be obtained. Smaller peptid e fragments, on the other hand, can provide much more detailed local structural information. Figur e 3-4 shows the peptide sequence mapping of the protein based on the data fr om the Figure 3-3. The pept ides found cover all the sequences of the protein. By comparing both larger and smaller residues (such as 1-29 vs. 1-11 and 70-106 vs. 87-106), more detailed local structural information can be obtained. The effect of digestion time is shown in Figures 3-5 and 3-6. Figure 3-5 shows the spectra of myoglobin in solution after one (top) and two (botto m) minute digestion times. The spectra showed the difference in terms of peak patterns, but 2 min digestion gave better absolute intensity. The 5 minute samp le (Figure 3-6) does not show better signal/noise, but the longer digestion time produced more pept ides, due to the limited digestion rate. The peptide coverages for different digesti on times are shown in Figures 3-7 and 3-8. The coverages are about the same for the 1, 2, and 5 min samples. However, when digestion time is longer (5 min), more short residues appear. This indicates that digestion time may be a factor that can be used to control t he extent of fr agmentation.

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39 Results from H/D exchange experiments on myoglobin in solution are presented in Figures 3-9 through 3-13. Some residues (Residue 1-29, for example) are the same as those examined by Buijs15, but smaller fragments are also analyzed. The general trends of mass shifts of is otopic distribution peaks are shown in Figure 3-9, which includes residues as fo llows: (a) 1-29, (b) 12-29, (c) 30-69, (d) 70106, (e) 107-137, (f) 138-153. As seen in Figu re 3-10, the centroid al masses for all the residues increased with incr easing H/D exchange time. W hen all the residues are compared (Figure 3-11), diffe rent mass shift patterns are observed. Residues 1-29 and 30-69 show more hydrogens exchanged than residues 70-106 and especially 107-137, which has a mass increase of less than 1 Da over the 100 minute exchange time. This finding is similar to Buijs work. Gilmans hin found that parts of the G and H helices (residue 101-148) in myoglobin48 form an extremely stable core49, that protects those hydrogens from being exchanged. This can ex plain the low mass increase of residue 107-137 and part of residue 70-106. In most ca ses, the mass distri bution width (Figure 3-12) also increased with exchan ge time, as observed by Buijs15, indicating that deuterium was incorporated into the peptide backbone in a more heterogeneous fashion with longer exchange time. Exchangeable hydrogens in proteins and peptides can be roughly classified into two groups, according to their relative reactivity toward H/D exchange. One group corresponds to hydrogens which are unprotec ted or barely protec ted, including side chain or terminal hydrogens. Some amide hydrogens may also be in this group due to their reactivity. Another group of hydrogens are protected and not easily exchanged. In Buijs work15, it was assumed that any hydrogens exchanged within 30 seconds belong

PAGE 40

40 to the first group. Based on this assumpti on, the mass increase after H/D exchange can be classified in 2 parts, as follows: )] (3-1) In equation 3-1, total mass increase is due to the exchange from both the first group (with m1 exchangeable hydrogens) and the second group (with m2 exchangeable hydrogens). kex is the exchange rate of the hy drogens in the second group; and is the deuterium percentage of all the hydrogens in the solvent. The difference between the total mass increase and the in crease from the first group, can be plotted versus the exchange time t The maximum mass increase of this difference, called the relative mass increase, should be at sufficient long exchange times. Based on this assumption, the relative mass changes of the 2 residues (1-29 and 3076) are plotted versus exchange time, as shown in Figure 3-13, which also shows curves simulated using the above formula. T he maximum relative mass change, i.e., the value of was assigned by assuming 2 factor s: (1) the mass increase almost reaches the maximum after 90 minutes of exchange; (2) is calculated by multiplying the percentage of deut erium (0.9) in the solvent and an integer, so that the product is close to the maximum mass incr ease from the experiment. The simulation curve from the reference15 was calculated based on t he data provided in their publication. The rate constants fit to the cu rrent work are greater than those in the reference. The average exchange rate c onstants for residue 1-29 and residue 30-69 are 4.50 h-1 and 5.52 h-1, respectively, compared to 0.726 h-1 and 4.09 h-1 obtained by Buijs. The maximum relative mass increases are sma ller than those reported in the reference.

PAGE 41

41 This apparent difference may come from back exchange or experim ental error due to the lack of sample replicates in this work. Binding Isotherms of Myoglobin on Nanoparticles The purpose of the experiments described in this section was to determine the maximum amount of myoglobin adsorbed on various nanoparticles. The adsorption was assumed to follow the Langmuir model50. Experimental Section Materials Nanoparticles used for this research were mainly characterized for nominal size, size distribution, and specific surface area. Size distributions of suspended particles were measured using a differential sedimentation CPS Disc CentrifugeTM (CPS Instruments, Stuart, FL). Samples were dispersed in deionized water and phosphate buffered saline (PBS) by 1 minute bath sonication and the resultant particle size distributions were measured by differentia l sedimentation. BET (Brunauer, Emmett and Teller) surface area was measured usi ng a Quantachrome Autosorb 1C-MS (Quantachrome Instruments, Boynton Beach, FL). The Co(core)/Co3O4 (shell) nanoparticles had a nominal size range of 5 20 nm (diameter), and the primary particle size distribution was determined to be 10.5 2.3 nm, with a specific surface ar ea (measured by BET) of 36.39 m2/g. Silver particles had a nominal size range of 20 30 nm (diameter), the primary particle size distribution was determined to be 26.6 8.8 nm, with a specific surfac e area (BET) of 14.53 m2/g. TiO2 had a nominal size of 25 nm (diameter), t he primary particle si ze distribution was determined to be 20.5 6.7 nm, with a specific surfac e area (BET) of 45.41 m2/g. Cobalt and silver nanoparticles were purc hased from Quantum S phere (Santa Ana, CA,

PAGE 42

42 USA). TiO2 was purchased from Degussa (Essen, Germany). Nickel nanoparticles with a nominal diameter of 50 nm and a spec ific surface area (BET) of 8.15 m2/g were obtained from Argonide Inc. (S anford, Florida). Colloidal Au particles with a nominal diameter of 20 nm were synthesized in the Particle Engineering Research Center by reduction of gold chloride with citrate. These par ticles had particle size distribution of 20 +/1 nm (diameter) and calculated s pecific surface area (BET) of 16 m2/g. Procedure for generation of protein adsorption isotherms Myoglobin solution from equine skeletal muscle (Sigma) and gold ,TiO2, nickel, silver or cobalt nanoparticles were vortex-mixed in 5 mM HEPES buffer at room temperature for 2 hours and centrifuged for 5 minutes at 18000 rpm (Sigma Centrifuge, Model 1-15). The concentrati on of free myoglobin (non-adso rbed) in the supernatant was determined by measuring the UV absorbanc e at 410 nm (Molecular Devices UV/vis spectrometer, Spectramax plus 384, Sunnyva le, CA). The amount of protein adsorbed on nanoparticles was obtained by subtracting t he free myoglobin from the initial amount. Results and Discussion The adsorption data were fit to theoretical curves, which were calculated based on the Langmuir adsorption model50: (3-2) In equation 3-2, Ab represents the amount of protein binding to nanoparticles, Amax is the maximum amount of protein binding to nanoparticles, Ab/Amax is the fraction of the particle surface covered, c is the free pr otein concentration, and k is the Langmuir adsorption constant.

PAGE 43

43 The adsorption isotherms are shown in Figure 3-14. The diamonds represent free protein in solution, and the triangles represent protein bound on nanoparticles. The adsorption generally follows the trend of the Langmuir model, with an asymptote at high concentration. It seems that myoglobin has the best adsorption on cobalt nanoparticles with the least amount of free protein in solution. Nickel is next to cobalt in terms of the adsorption amount. It can be seen that the size of the nanopar ticle and the adsorption maximum dont follow a simple relationship, indicating that particle size is not the only factor affecting the amount adsorbed. In fact, nanoparticles tend to aggregate when suspended in solution. This aggregation increases the size of nanoparticles51 and decreases the solvent accessible surface area. Other surface characteristi cs, such as charge, polarity and composition, may all contribute to the ad sorption. Interactions between the protein and the adsorbent, such as electrostatic in teractions, hydrophobic interactions, and specific chemical interactions also play important roles4. The mechanism of selective adsorption of proteins on various adsorbents has been attributed to electrostatic interactions52. Mass spectrometric methods were tried for protein adsorbed on each of these nanoparticles. It was found that nickel and silv er nanoparticles were better than all others in terms of the mass spectral intens ity. For other nanoparti cles, the adsorption may be so strong that, even after digestion peptides are not released into solution. Based on the adsorption result s, silver and nickel nanoparti cles were chosen as the adsorption substrate for furt her MS analysis in the followin g two sections. The weight

PAGE 44

44 ratio of protein to nanoparticles was set to 1:40 to achieve the maximum adsorption and strong MS signals. Pepsin Digestion In this section, pepsin was used as the enzymatic protease to digest protein in solution and adsorbed on silver and nickel nanoparticles. Mass spectra were obtained on a 4.7 T FTICR mass spectrometer. The objective was to identify digested myoglobin in the above two cases before carry ing out H/D exc hange experiments. Experimental Section Materials Myoglobin from equine skeletal muscle was purchased from Sigma Aldrich. Porcine gastric pepsin A used as a pr otease was purchased from Biochemical Corporation. 5 mM HEPES solution (Fisher Biotech) was used as the buffer, pH 6.5. Cobalt, nickel and silver nanoparticles were purchased from QuantumSphere. Instrument The instrument used for this study was Bruker 4.7 T FTICR mass spectrometer (Bruker Daltonics, Billerica, MA, USA) in the positive ion mode53. Samples were introduced and ionized using an Analytica of Br anford electrospray ionization source, with a heated metal capillary (HMC) and a hexapo le trapping region. No nebulizing gas was needed in the source. All the samples were directly infused into the ionization source region at a flow rate of 15 L /hr. The mass range scanned was from m/z 200 to 1500 Da with accumulation of 25 to 100 scans. Ions were produced by the application of a high voltage (~1.5-2.0 kV) to a metal union in contact with the sample solution. The charged solution was pumped toward the HM C, maintained around + 140 V and 120C. Ions were trapped and accumulated in the hexapole region. After tuning for optimal

PAGE 45

45 transmittance, ions were pulsed toward t he ICR cell. Detection was accomplished by collecting the induced image current for 256 K data point transients. After Fourier transformation, the time-domain image cu rrent provided the mass spectrum. Analysis of protein di gested with pepsin In solution. Ten L of 50 mM myoglobin in 5 mM HEPES was mixed with 65 L of 6 mM porcine gastric pepsin in 6% acetic acid at 0 for 2 minutes. The solution was desalted with C18 ZipTips (Millipore) and eluted with 0.1% formic acid in 80:20 acetonitrile:H2O. The eluate was infused into a 4.7 T FTICR mass spectrometer. On Ag or Ni nanoparticles. Myoglobin and nanoparticles (1:40) were dissolved/suspended in 5 mM HEPES buffer for 2 hours. The particle part was separated, washed, and diges ted with pepsin. The supernatant was separated after centrifugation, desalted, and infused into a 4.7 T FTICR mass spectrometer. Results and Discussion Mass spectra were obtained after pepsin dige stion for myoglobin both in solution and adsorbed on nanoparticles. Peptides in t he protein sequence were identified, and the coverage was obtained bas ed on sequence mapping. Figure 3-15 shows the spectr a of the digest in solution and adsorbed on nickel and silver nanoparticles. As can be seen, the general pattern of the spectra is similar. But more small peaks are observed for myoglobin on nanoparticles. For the solution sample, the most intense peak is from the residue 71-107. In the spectrum for myogl obin on nickel nanoparticles the 71-107 residue is again the most intense. More small peaks appear especially in the low mass range below 500. Some fragments, such as residue 71-87 and 59-102, which are not significant in

PAGE 46

46 the spectrum for protein in solution, were identified here. This difference indicates that the adsorption of protein on nanoparticles may affect the digestion pattern or extent. In the spectrum for myoglob in on silver nanoparticles, there are many small peaks (for example, residues 71-77, 71-87, 59-102), compared to the spec trum of myoglobin in solution. This is similar to the spectr um for protein adsorbed on nickel particles. The 71-107 residue peak is very intense, but the most intense peak corresponds to the 139154 residue. Fig. 3-16 compares peptide coverage of my oglobin in solution, adsorbed on Ni and adsorbed on Ag after 2 minutes digestion by pepsin. The sequence coverage for myoglobin in solution was 95% while the coverages for myoglobin bound to Ni and Ag nanoparticles were 93% and 87%, respectively. The sequence maps are similar for the three cases. But there are also different fragments observed for myoglobin adsorbed on nanoparticles, in particular, residues 59-102 and 71-87. These results indicate that adsorption on nanoparticles may change the fragmentation mode of protein upon pepsin digestion. Protease XIII Digestion In this section, protease type XIII wa s used to digest protein in solution and adsorbed on silver nanoparticles. Mass spec tra were obtained on a 14.5 T FTICR mass spectrometer. The objective was to identif y digested myoglobin in the above two cases before carrying out H/D exchange experiments. Experimental Section Materials Apomyoglobin from equine skeletal muscl e and protease XIII from Aspergillus saitoi were purchased from Sigma Aldrich. Silver nanopart icles were purchased from

PAGE 47

47 QuantumSphere. 20 nm alumina membrane syringe filters were purchased from Whatman. Instrument The instrument used for this study was a hybrid LTQ 14.5 T FTICR Mass Spectrometer (Thermo Electron Corp., San Jose, CA) custom-built at the National High Magnetic Field Laboratory (NHMFL)54. The instrument was operat ed in the positive ion mode, and the mass range scanned was from m/z 400 to 1600 Da. Analysis of protein dig ested with protease XIII In solution. A 50 L aliquot of 8 pmol/ L apomyoglobin (0.136 mg/mL) in 50mM sodium phosphate buffer, pH 7.8, was mixed with 50 L of 1.492mg/mL protease XIII in 1.0% formic acid at 0 for 2 minutes. The mixture was desalted and analyzed with the 14.5 T FTICR mass spectrom eter at the NHMFL. On Ag nanoparticles. A 100 L aliquot of 80 pmol/ L apomyoglobin (1.36 mg/mL) in 50mM sodium phosphate buffer, pH 7.8, was added to 2.72 mg of Ag nanoparticles (20-30 nm, Quantum Sphere) and rotated for 5 hrs. The particle part was separated, washed and digested by 500 L of 1.492 mg/mL protease XIII solution in 1.0% formic acid at 0 and vortex-mixed for 2 minutes to prevent precipitation. The supernatant was separated with 20 nm syringe filters, desa lted, and infused into the 14.5 T FTICR mass spectrom eter at the NHMFL. Results and Discussion Figure 3-17 shows the mass spectrum with a charge state distribution of intact apomyoglobin in solution. Analysis of the change of the charge state distribution may help study the conformational ch anges of protein; however, th is is not the focus of the research.

PAGE 48

48 The peptide coverage and s equence mapping shown in Figure 3-18 correspond to apomyoglobin in solution and adsorbed on Ag nanoparticles with 2 min digestion using protease XIII. The sequence coverage for apomy oglobin in solution is 85.6% while that for apomyoglobin bound to Ag nanoparticles is 84.3%. This coverage is about the same as seen in the previous section, but pepsin seems to give a somewhat higher coverage. By using protease type XIII, however, very small fragments containing as few as 2 amino acid residues (such as residue 104-105) can be obtained. This will be beneficial when studying local structural stability, because it will be possible to obtain structural information at the several amino acid residue resolution level. The digestion pattern is also different than that observed in peps in digested samples. Combining these 2 proteases should be very helpful in elucidating protein structure. Other advantages of using protease XIII include increased elec trospray ionization efficiency for mass spectrometric detection, increased signal to noise ratio, and lower tendency of selfdigestion of the protease18. The common fragments for protein in solution and on silver correspond to a coverage of 74.5% (Figure 3-19). This implies that it is very promising to study globally, as well as locally, the conformational changes of myoglobin adsorbed on nanoparticles by comparing the mass spectrometric data for these common fragments. Summary As a summary of this chapter, t he following statements can be made: H/D exchange of myoglobin in soluti on was performed and results were compared with reference data; Binding isotherms of myoglobin ads orbed on nanoparticles were obtained; Myoglobin in solution was digested with pepsin and protease XIII and peptide sequences were mapped;

PAGE 49

49 Myoglobin adsorbed on nanoparticles (Ni, Ag) was digested with pepsin and protease XIII and peptide sequences were mapped; Protease XIII provides different fragm entation patterns for myoglobin than pepsin, suggesting a promising way of studying both global and local structure of myoglobin by mass spectrometry.

PAGE 50

50 Figure 3-1. Mass spectra of myoglobin in solution obtained with ES I-Q-TOF: top: 21 M; bottom: 3.6 M

PAGE 51

51 Figure 3-2. Expansion of 464 to 476 m/z region of Figure 3-1

PAGE 52

52 Figure 3-3. Mass spectrum of 3.6 M myoglobin in solution, th e same as the bottom of Figure 3-1, with labeled re sidues. *: For residue 70-106, smaller residues of it (residue 70-86 and 87-106) can be used to study the structure of the peptide in higher resolution Res 30-69 Res 70-106 Res 138-153 Res 1-29 Res107-134 Res 107-137 Res 70-86 Res 87-106

PAGE 53

53 Figure 3-4. Peptide co verage obtained with 3.6 M concentration of myoglobin 1 11 21 31 41 51 61 71 A-Helix B-Helix C-Helix D-Helix E-Helix GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG81 91 101 111 121 13 1 141 151F-Helix G-Helix H-HelixHHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG

PAGE 54

54 Figure 3-5. Mass spectra of myoglobin in solution obtained after different digestion times: top: 1 minute; bottom: 2 minutes 1 min 2 min

PAGE 55

55 Figure 3-6. Mass spectra of myoglobin in solution obtained after different digestion times: top: 5 minutes; bottom: 2 minutes 5 min 2 min

PAGE 56

56 Figure 3-7. Comparison of peptide coverage after different digestion times: top: 1 minute; bottom: 2 minutes 1 11 21 31 41 51 61 71 A-Helix B-Helix C-Helix D-Helix E-Helix GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG81 91 101 111 121 131 141 151F-Helix G-Helix H-HelixHHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG 1 min digestion 2 min digestion

PAGE 57

57 Figure 3-8. Comparison of peptide coverage after different digestion times: top: 5 minutes; bottom: 2 minutes 1 11 21 31 41 51 61 71 A-Helix B-Helix C-Helix D-Helix E-Helix GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG81 91 101 111 121 13 1 141 151F-Helix G-Helix H-HelixHHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG 5min digestion 2 min digestion

PAGE 58

58 Figure 3-9. Mass spectra showin g mass shifts during H/D exchange Before H/D Exchange Before H/D Exchange 30s 30s30s 5 min 5 min 20 min 20 min 90 min 90 min 20 hr Before H/D Exchange Before H/D Exchange 5 min 5 min 20 min 20 min 90 min 90 min 20 hr20 hr Before H/D Exchange Before H/D Exchange 30s 30s 5 min 5 min 90 min 20 min 20 min 90 min 20 hrRes. 1 29 Res. 12 29 Res. 30 69 Res. 70 106 Res. 107 137 Res. 138 153

PAGE 59

59 Figure 3-10. Deuterium uptake of various residues in myoglobin in solution 8.0 9.0 10.0 11.0 12.0 13.0 14.0 0.1 1.0 10.0 100.0Deuterium Uptake (Da)Exchange Time (min)Res. 1 29 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 0.11.010.0100.0Deuterium Uptake (Da)Exchange Time (min)Res. 12 29 10.0 11.0 12.0 13.0 14.0 15.0 16.0 0.1 1.0 10.0 100.0Deuterium Uptake (Da)Exchange Time (min)Res. 30 69 9.0 9.2 9.4 9.6 9.8 10.0 10.2 0.10 1.00 10.00 100.00Deuterium Uptake (Da)Exchange Time (min)Res. 70 106 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 0.11.010.0100.0Deuterium Uptake (Da)Exchange Time (min)Res. 107 137 5.0 5.5 6.0 6.5 7.0 7.5 0.11.010.0100.0Deuterium Uptake (Da)Exchange Time (min)Res. 138 153

PAGE 60

60 Figure 3-11. Comparison of mass increase for 6 residues 4.0 6.0 8.0 10.0 12.0 14.0 16.0 0.11.010.0100.0Deuterium Uptake (Da)Exchange Time Res. 1 29 Res. 12 29 Res. 30 69 Res. 70 106 Res. 107 137 Res. 138 153

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61 Figure 3-12. Comparison of the c hange of peak width in H/D exchange 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 0.11.010.0100.0Peak Width (Da)Exchange Time (min) residue 1-29 residue 12-29 residue 30-69 residue 70-106 residue 107-137 residue 138-153

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62 Figure 3-13. Mass increase simulation for residue 1-29 and 30-69 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0.10 1.0010.00100.001000.00Deuterium Uptake (Da)Exchange Time (min)Res. 1 29 Experiment Simulation BuijsSimulation 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0.101.0010.00100.00Deuterium Uptake (Da)Exchange Time (min)Res. 30 69 Experiment Simulation Buijs Simulation

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63 Figure 3-14. Binding isotherms of myoglobin on nanoparticles 0 50 100 150 200 250 300 050100150200250300350 Initial Protein Amount (g)Final Protein Amount (g) Bound Protein Free Protein 0 200 400 600 800 1000 020040060080010001200 Initial Protein Amount (g)Final Protein Amount (g) Bound Protein Free Protein 0 200 400 600 800 1000 020040060080010001200 Initial Protein Amount (g)Final Protein Amount (g) Bound Protein Free Protein 0 200 400 600 800 1000 020040060080010001200 Initial Protein Amount (g)Final Protein Amount (g) Bound Protein Free ProteinAu, 20 nm Ni, 50 nm TiO2, 25 nm Co/Co3O4, 5 20 nm 0 0.1 0.2 0.3 0.4 0.5 0.6 00.20.40.60.8Final Protein Amount (mg)Initial Protein Amount (mg)Ag, 20 30 nm Bound Protein Free Protein

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64 Figure 3-15. Mass spectra of myoglobin digest in solution and on nanoparticles: top: myoglobin in solution; medium: myoglobin binding to nickel; bottom: myoglobin binding to Ag Myoglobin on Ni 200400600800100012001400 0.0 5.0e+6 1.0e+7 1.5e+7 2.0e+7 2.5e+7 Myoglobin on Agm/z 200400600800100012001400 0.0 2.0e+6 4.0e+6 6.0e+6 8.0e+6 1.0e+7 1.2e+7 Myoglobin in Solution 200400600800100012001400 0 2e+7 4e+7 6e+7 8e+7 1e+8 Res. 71 107 Res. 139 154 Res. 31 70 Res. 71 107 Res. 31 70 Res. 34 70 Res. 71 107 Res. 108 138 Res. 13 30 Res. 71 107 Res. 71 107 Res. 139 154 Res. 71 107 Res. 108 138 Res. 59 102 Res. 31 70 Res. 34 70 Res. 138 153 Res. 71 87 Res. 31 70 Res. 71 107 Res. 139 154 Res. 71 107 Res. 108 138 Res. 59 102 Res. 34 70 Res. 71 87 Res. 31 70 Res. 71 107 Res. 71 77 Res. 45 58

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65 Figure 3-16. Sequence mapping of myoglobin in solution and on nanoparticles, after pepsin digestion 231 11 21 31 41 51 61 71A-Helix B-Helix C-Helix D-Helix E-HelixGLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG81 91 101 111 121 131 141 15181F-Helix G-Helix H-HelixHHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG In solution On Ni On Ag .z

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66 Figure 3-17. Mass spectrum of intact apomyoglobin 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 848.56 z=20 893.11 z=19 808.15 z=21 942.73 z=18 771.51 z=22 998.12 z=17 1060.44 738.01 z=23 1131.01 z=15 853.41 898.37 812.91 707.26 948.23 1211.79 1003.89 1066.51 1304.85 775.96 1137.54 1218.79 1312.39 858.40 903.48 953.56 983.38 1009.65 817.53 1072.57 1225.65 1144.07 1359.70 1109.59 z=16

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67 Figure 3-18. Sequence mapping of myoglobin in solution and on silver nanoparticles digested by protease XIII M GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG81 91 101 111 121 131 141 151HHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG In solution On Ag1 11 21 31 41 51 61 71 A-Helix B-Helix C-Helix D-Helix E -Helix F-Helix G-Helix H-Helix

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68 Figure 3-19. Common fragm ents of myoglobin in solution and on nanoparticles digested by protease XIII

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69 CHAPTER 4 H/D EXCHANGE MASS SPECTROMETRY OF MYOGLOBIN ON SILVER NANOPARTICLES To achieve the initial goal of investi gating protein conformational change during adsorption on nanoparticles, efforts were ma de to conduct HDEX-MS experiments for myoglobin on silver nanoparticles. Initially, myoglobin samples (in soluti on and on silver nanoparticles) after H/D exchange were prepared and sent to the National High M agnetic Field Laboratory (NHMFL) at Florida State University (FSU ). Unfortunately, the analysis by HPLC ESI FTICR encountered some unknown contamination from the samples and the analysis was discontinued. Additional experiments were carried out to seek t he reasons for the sample contamination. It was found that the filters used to remove the nanoparticles not only adsorbed the digested peptides, but also contam inated the filtrate. Alternative filters were used to eliminate the contamination issue. Recently, myoglobin samples (in soluti on and on silver nanoparticles) after H/D exchange were prepared and analyzed using an LTQ-Orbitrap at the University of South Florida (USF). Analysis for Sample Contamination Source Sample Preparation for HDEX-MS Materials Myoglobin from equine skeletal muscle and protease XIII from Aspergillus saitoi were purchased from Sigma Aldr ich; heavy water was 99.9% D2O (Sigma Aldrich); 20 nm alumina membrane syringe filters were purchased from Whatman; 200 nm nylon syringe filters were purchased from Fisher; 200 nm Supor membrane syringe filters

PAGE 70

70 were purchased from Pall Gelman Laboratory; Blue plus 2 molecular weight markers were purchased from Invitrogen. Instrument The instrument used for this study was a hybrid LTQ 14.5 T FTICR Mass Spectrometer (Thermo Electron Corp., San Jose CA) custom-built at the National High Magnetic Field Laboratory (NHMFL), also de scribed in Chapter 3. The instrument was operated in the positive ion mode, and t he mass range scanned was from m/z 400 to 1600 Da. H/D exchange mass spectrometry of protein in solution Aliquots of 10 L of 80 pmol/ L myoglobin in 50mM sodium phosphate buffer, pH 7.8, were each mixed with 90 L of 50mM sodium phosphate in D2O (99.9%, Sigma) buffer, then incubated for different lengths of time to allow for H/D exchange. The sample was then digested with 100 L of 1.492 mg/mL protease XIII solution in 1.0% formic acid at 0 for 2 minutes, filtered with a 20 nm nanofilter, frozen in liquid nitrogen, and stored overnight. The samples were thawed and infused into the 14.5 T FTICR mass spectrometer at t he NHMFL immediately. H/D exchange mass spectrometry of protein on Ag nanoparticles 100 L of 80 pmol/ L apomyoglobin in 50mM sodium phosphate buffer, pH 7.8, was added to 2.72 mg of Ag nanoparticles ( 20-30 nm, QuantumSphere) and rotated for 5 hrs. The particle part was separated and washed. 10 L aliquot of the particle suspension was mixed with 90 L D2O and incubated for different lengths of time to allow for H/D exchange. The samp le was then digested with 100 L of 1.492 mg/mL protease XIII solution in 1.0% formic acid at 0 for 2 minutes, filtered with a 20 nm

PAGE 71

71 nanofilter, frozen in liquid nitrogen and stored overnight. The samples were thawed and infused into the 14.5 T FTICR mass spectr ometer at the NHM FL immediately. The spectra indicated the presence of a co ntaminant with very fe w peptide signals. The source of the cont amination was investigat ed as described below. Gel electrophoresis analysis for sample contamination Aliquots of samples (Figur e 4-1) were resolved on a 4-12% Bis-Tris-SDS-PAGE gel. Blue plus 2 was used as a standard. Pure myoglobin and protease XIII were analyzed as blank controls. After electrophores is, the gel was fixed in 50% methanol and 7% acetic acid for 30 minutes twice, st ained with Sypro Ruby gel stain overnight, washed with 10% methanol and 7% acetic acid for 30 minutes, then washed with H2O twice. The gel was analyzed with a UV trans-illuminator (Bio-Rad laboratories, Universal Hood II, CA). Results and Discussion In an effort to understand why the samples previously sent to the NHMFL appeared to have poor digestion efficiency, SDS-PAGE analyses were conducted on newly digested samples as well as several samples sent to the NHMFL. The samples included 11 freshly made digests and 2 samples sent to the NHMFL. Figure 4-1 shows the image of the digested myoglobin samples. Samples in lanes 2-5 were not filtered. Samples in lanes 6-14 were filtered with a 20 nm filter. Samples were separated on 4-12% Bis-Tris gel and stained with Sypro Ruby. Sypro Ruby protein gel stain is a sensit ive fluorescent stain that is used in polyacrylamide gel electrophoresis (PAGE) to detect separated proteins. It has a detection limit of ~ 1ng/band, so there appeared to be very little undigested protein in any of these samples. However, there appear ed to be very little protein of any type in

PAGE 72

72 the samples, as bands corresponding to protease XIII were also absent. This was surprising, so another experiment was condu cted to evaluate the digestion of myoglobin over time, as well as the impact of 20 nm filtration on the samples. Samples were analyzed on 4-12% Bis-Tris gel with Sypro Ruby staining. The image is shown in Figure 4-2. From these results, two things are appar ent. Protease XIII digestion for 2 minutes appears to digest well over 95% of intact my oglobin. However, 20 nm filtration causes dramatic loss of both myogl obin and protease XIII, likely accounting for the results observed above. Because peptides could not be observed in this analysis, it was unclear how filtration was affe cting peptide concentrations, t hough it is likely that the peptides were also significantly reduced. It is necessary to remove nanoparticles pr ior to LC analysis to avoid plugging the column. However, it was shown from the PAG E analysis that the 20 nm filters were not appropriate for preparing samples that would subsequently be analyzed for proteins and peptides. Because significant aggregation of s ilver particles occurs under conditions of myoglobin binding, 200 nm nylon filters were next in vestigated (Figure 4-3). These results demonstrated that 200 nm nylon filtration produced much better protein yields. However, when digests we re analyzed by MS, high concentrations of contaminants were identified. These could not be removed by washing the filter with aqueous or organic solvents as evident in Figure 4-4, which shows the MS spectra obtained from myoglobin digests filt ered with 200 nm nylon filters. Analysis of the blank sample presented in Figure 4-4 indicates the presence of strong peaks at 679.4, 701.4 and 717.4 (m/z) in t he blank sample after filtration. These

PAGE 73

73 peaks do not appear in the solution without filt ration, suggesting that the contamination originated from the nylon filt er. These are more evident on the bottom panel of Figure 45. The m/z range on the top is from 200 to 1600, and on the bottom is from 678 to 724, which shows an expansion of the m/z region. Because of the contamination found in nylon filters, alter nate filter materials were investigated. It was found that 200 nm S upor membranes did not adsorb sample and did not leach contaminants. These filters were used to conduct an abbreviated H/D exchange experiment, in which samples were analyzed by direct infusion of digests on an LTQ-Orbitrap at USF. As evidenced in the spectra in Figures 4-6 through 4-7, large numbers of peptides were present in these samples, although some of the peptides were quite large (>3500amu), which may indicate incomplete digestion. Figure 4-6 provides an example of the spectra collect ed by direct infusion of myoglobin digests. To ensure that enzymatic activity was ma ximal, a new bottle of Protease XIII was purchased and digestion effectiveness was agai n determined by SDS-PAGE (Figure 48). From Figure 4-8, it is apparent that the new protease XIII enzyme also removes >95% of intact myoglobin after 2 minutes dige stion. These results seem very similar to those obtained with the Protease XIII used in pr ior experiments. Follo wing filtration with 200 nm Supor membranes (lanes 10-13), very li ttle intact myoglobin is visible while the protease XIII concentration appears unaffected.

PAGE 74

74 HDEX-MS of Myoglobin using LTQ-Orbitrap Experimental Section Materials Myoglobin from equine skeletal muscle and protease XIII from Aspergillus saitoi were purchased from Sigma Aldr ich; heavy water was 99.9% D2O (Sigma Aldrich); 200 nm Supor membrane syringe filters were purchased from Pall Gelman Laboratory. Instrument The instrument used for this study wa s an LTQ-Orbitrap mass spectrometer (Thermo Electron Corp., San Jose, CA) at the University of South Florida. The instrument was run in the positive ion mode, with the m/z range scanned from 235 to 2000 Da. Analysis of protein dig ested with protease XIII In solution : a 100 L aliquot of 16 pmol/ L myoglobin (0.272 mg/mL, equine skeletal, Sigma) in 10 mM ammonium acet ate buffer, pH 7.2, was mixed with 100 L of 2.98 mg/mL protease XIII (Aspergillus saitoi Sigma) in 1.0% formic acid at 0 for 2 minutes. The solution was filtered with a 200 nm Supor filter, and immediately frozen in liquid nitrogen. Then 200 L ice-cold acetonitrile was added to the frozen sample, which was directly infused into an LTQ-Orbitrap mass spectrometer at USF. On Ag nanoparticles: a 100 L aliquot of 16 pmol/ L apomyoglobin (0.272 mg/mL) in 10 mM ammonium acetate buffer, pH 7.2, was added to 5.44 mg of Ag nanoparticles (20-30 nm, Quantum Sphere) and rotated for 5 hrs. The particle part was separated, washed and digested with 100 L of 2.98 mg/mL protease XIII solution in 1.0% formic acid at 0 and vortex-mixed for 2 minutes to prevent precipitation. The suspension was filtered with a 200 nm Supor filter, and frozen in liquid nitrogen

PAGE 75

75 immediately. Then, 200 L ice-cold acetonitrile was added to the frozen sample, which was directly infused into an LTQ-Orbitrap mass spectrometer at USF. H/D Exchange mass spectrometry of protein on Ag nanoparticles In solution : Aliquots of 10 L of 160 pmol/ L myoglobin in 10 mM ammonium acetate buffer, pH 7.2, were each mixed with 90 L of 10 mM ammonium acetate D2O buffer, and incubated for different lengths of ti me to allow for H/D exchange (0, 1, 4, 15, and 60 minutes). The samples were digested with 100 L of 2.98 mg/mL protease XIII (Aspergillus saitoi, Sigma) in 1.0% formic acid at 0 for 2 minutes. The solution was filtered through a 200 nm Supor filter, and im mediately frozen in liquid nitrogen. Then 200 L ice-cold acetonitrile was added to the frozen sample, which was directly infused into an LTQ-Orbitrap mass spectrometer at USF. On Ag nanoparticles : A 100 L aliquot of 16 pmol/ L myoglobin (0.272 mg/mL) in 10 mM ammonium acetate bu ffer, pH 7.2, was added to 5.44 mg of Ag nanoparticles (20-30 nm, QuantumSphere) and rotated for 5 hrs. The par ticle part was separated, washed, and a 10 L suspension including most of the particles was mixed with 90 L of 10 mM ammonium acetate D2O buffer. The sample was incubated for different lengths of time to allow for H/D exchange (0, 1, 4, 15, and 60 minutes), digested with 100 L of 2.98 mg/mL protease XIII soluti on in 1.0% formic acid at 0 and vortex-mixed for 2 minutes to prevent precipitation. T he suspension was filtered with a 200 nm Supor filter, and immediately frozen in liquid nitrogen. Then 100 L ice-cold acetonitrile was added to the frozen sample, which was direct ly infused into an LTQ-Orbitrap mass spectrometer at USF.

PAGE 76

76 Results and Discussion The peptide sequence mapping based on the results from the LT Q-Orbitrap is shown in Figure 4-9, together with previous mapping results from data obtained at the NHMFL for comparison. The spectra from Or bitrap data are very complex and hard to analyze in full scan mode, but many commo n fragments are observed when compared with the NHMFL data. The peptide coverages were almost the same in both cases, indicating that this procedure is a promising way to achieve the goals of this research. The H/D exchange results (Figure 4-10) fr om the Orbitrap spectra show a mass shift during H/D exchange, even though in the solution samples, the mass increase is not very evident. However, all the residues showed a significant mass increase when adsorbed on silver nanoparticles. The mass in crease can be better seen in Figure 4-11, which shows the deuterium atom uptake for residues 12-18, 39-48, and 32-42. During the H/D exchange, the general trend is that mass increases wit h the exchange time, but there are some fluctuations. More replicates are necessary to investigate this problem. Also, back exchange may be serious during the detection process. Care must be taken to address this issue. On the other hand, Fi gure 4-11 shows that there were significant mass increases from H/D exchange when myoglobin was adsorbed on nanoparticles. Deuterium uptake at 4 minutes for all the residues was much greater than that in solution, even after the exchange was performed in solution for more than 60 minutes. This preliminary result on the relative ma ss increases between the solution and on the nanoparticles is encouraging, since it clearly shows that upon adsorption on silver nanoparticles, significant conformational change occurred, resulting in greater deuterium uptake. It will be very interesting to investigate further how the myoglobin, or

PAGE 77

77 other proteins on silver or other nanoparticles change their global or local structure by the method developed here.

PAGE 78

78 Figure 4-1. SDS-PAGE of freshly-made my oglobin digests and old samples. Samples 2,3 were prepared without filtration; other s were prepared following filtration with 20 nm Alumina filter. 1) Bl ue plus 2 standard marker, 2) 5 L myoglobin 80 pmol/ L + 45 L Phosphate buffer + 50 L 1% Formic acid, 3) 10 l myoglobin 80 pmol/ L + 40 L Phosphate buffer + 50 L 1% Formic acid, 4) 50 L protease XIII 1.4 mg/mL in 1% formic acid + 50 L phosphate buffer, 5) 50 L protease XIII 2.8 mg/mL in 1% formic acid + 50 L phosphate buffer, 6)-14) digestion 2 minutes, 6) 5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 2.8 mg/mL in 1% formic acid, 7) 10 L myoglobin 80 pmol/ L + 40 L phosphate buffer + 50 L protease XIII 2.8 mg/mL in 1% formic acid, 8)-10) & 12) 5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 1.4 mg/mL in 1% formic acid, 11) 10 L myoglobin 80 pmol/ L + 40 L phosphate buffer + 50 L protease XIII 1.4 mg/mL in 1% formic acid, 13) sample to the NHMFL (5 L myoglobin 80 pmol/ L + 45 L D2O phosphate buffer for one hour, then add 50 L protease XIII 1.4 mg/mL in 1% formic acid), 14) sample to the NHMFL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 1.4 mg/mL in 1% formic acid), 15) Blue plus 2 standard marker Myoglobin Protease XIII

PAGE 79

79 Figure 4-2. SDS-PAGE of fr eshly-made myoglobin digests. Samples 2-9, 12, 13 were prepared without filtration; others were prepared followi ng filtration with 20 nm Alumina filter. 1) Blue plus 2 standard marker; 2) digestion 0 minute, final protease XIII 0.7mg/mL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 1.4 mg/mL in 1% form ic acid); 3) digestion 0 minute, final protease XIII 1.4mg/mL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 2.8 mg/mL in 1% formic acid); 4) digestion 1 minute, final protease XIII 0.7mg/mL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 1.4 mg/mL in 1% formic acid); 5) digestion 1 minute, final pr otease XIII 1.4mg/mL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 2.8 mg/mL in 1% formic acid): 6) digestion 2 minutes, final protease XIII 0.7mg/mL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 1.4 mg/mL in 1% formic acid); 7) digestion 2 minutes final protease XIII 1.4mg/mL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 2.8 mg/mL in 1% formic acid); 8) digestion 5 minutes, final protease XIII 0.7mg/mL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 1.4 mg/mL in 1% formic acid); 9) digestion 5 minutes, final protease XIII 1.4mg/mL (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 2.8 mg/mL in 1% form ic acid); 10) myoglobin blank after filtration (5 L myoglobin 80 pmol/ L + 45 L Phosphat e buffer + 50 L 1% formic acid); 11) protease X III blank after filtration (50 L protease XIII 1.4 mg/mL in 1% formic acid + 50 L phosphate buffer); 12) myoglobin blank without filtration (5 L myoglobin 80 pmol/ L + 45 L Phosphate buffer + 50 L 1% formic acid); 13) prot ease XIII without filtration (50 L protease XIII 1.4 mg/mL in 1% formic acid + 50 L phosphate buffer); 14) digestion 2 minutes, final protease XIII 0.7m g/mL, after filtration (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 1.4 mg/mL in 1% formic acid); 15) digestion 2 minutes, final protease X III 1.4mg/mL, afte r filtration (5 L myoglobin 80 pmol/ L + 45 L phosphate buffer + 50 L protease XIII 2.8 mg/mL in 1% formic acid). M y o g lobin Protease XIII

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80 Figure 4-3. SDS-PAGE of freshly-made myoglobin digests. Samples 4-14 were prepared following filtration with 200 nm nylon filter 1) digestion 2 minutes, final protease XIII 0.7m g/mL, myoglobin 0.07mg/mL, without filtration; 2) digestion 2 minutes, final protease XIII 1.4mg/mL, myoglobin 0.07mg/mL, without filtration; 3) digestion 2 minut es, final protease XIII 1.4mg/mL, myoglobin 0.14mg/mL, without filtration; 4) digestion 0 minute, final protease XIII 0.7mg/mL, myoglobin 0.07mg/mL; 5) digestion 0 minute, final protease XIII 1.4mg/mL, myoglobin 0.14mg/mL; 6) digestion 1 minute, final protease XIII 0.7mg/mL, myoglobin 0.07mg/mL; 7) digestion 1 minute, final protease XIII 1.4mg/mL, myoglobin 0.07mg/mL; 8) digestion 1 minute, final protease XIII 1.4mg/mL, myoglobin 0.14mg/mL; 9) digestion 2 minutes, final protease XIII 0.7mg/mL, myoglobin 0.07mg/mL; 10) myoglobin after filtration (10 L myoglobin 80 pmol/ L + 40 L Phosphate buffer); 11) digestion 2 minutes, final protease XIII 1.4m g/mL, myoglobin 0.14mg/mL; 12) digestion 5 minutes, final protease XIII 0.7m g/mL, myoglobin 0.07mg/mL; 13) digestion 5 minutes, final protease XIII 1.4m g/mL, myoglobin 0.07mg/mL; 14) digestion 5 minutes, final protease XIII 1.4m g/mL, myoglobin 0.14mg/mL; 15) digestion 0 minute, final protease XIII 0.7mg/mL, myoglob in 0.07mg/mL, without filtration. Myoglobin Protease XIII

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81 Figure 4-4. Mass spectra of myoglobin digests and blank following filtration with 200 nm nylon filter Contaminant from nylon filter Peptides Contaminant from nylon filter Myoglobin digest filtered through 200 nm nylon filter 200nm nylon filter blank

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82 Figure 4-5. Mass spectrum of cont aminants from 200 nm nylon filter

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83 Figure 4-6. Mass spectrum of myoglobin digests following filtration with 200 nm Supor membrane

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84 Figure 4-7. Zoomed-in mass spectra of my oglobin digests following filtration with 200 nm Supor membrane

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85 Figure 4-8. SDS-PAGE of myoglobin digested with protease XIII after 200 nm Supor membrane filtration. Samples 1-9 and14-15 are prepared without filtration, samples 10-13 are filtered with 200nm Supor filter. 1) digestion 0 minute, final protease XIII 0.7mg/mL, myoglobin 0. 07mg/mL in low adhesion tube; 2) digestion 0 minute, final protease X III 0.7mg/mL, myoglobin 0.07mg/mL in regular tube; 3) digestion 1 minute, fi nal protease XIII 0.7mg/mL, myoglobin 0.07mg/mL; 4) digestion 1 minute, final protease XIII 1.4mg/mL, myoglobin 0.07mg/mL; 5) digestion 2 minutes, fi nal protease XIII 0.7mg/mL, myoglobin 0.07mg/mL; 6) digestion 2 minutes, fi nal protease XIII 1.4mg/mL, myoglobin 0.07mg/mL; 7) digestion 5 minutes, fi nal protease XIII 0.7mg/mL, myoglobin 0.07mg/mL; 8) digestion 5 minutes, fi nal protease XIII 1.4mg/mL, myoglobin 0.07mg/mL; 9) digestion 5 minutes, fi nal protease XIII 1.4mg/mL, myoglobin 0.07mg/mL; 10) digestion 2 minutes, fi nal protease XIII 0.7mg/mL, after filtration; 11) digestion 2 minutes, final protease XIII 1.4mg/mL, after filtration; 12) digestion 2 minutes, final protease XIII 0.7mg/mL, after filtration; 13) digestion 2 minutes, final protease XIII 1. 4mg/mL, after filtra tion; 14) digestion 2 minutes, final protease XIII 0.7mg/ mL, myoglobin 0.07mg/mL in regular tube; 15) digestion 2 minutes, final protease XIII 1.4mg/mL, myoglobin 0.07mg/mL in regular tube. Myoglobin Protease XIII

PAGE 86

86 Figure 4-9. Peptide co verage of myoglobin diges ted with protease XIII

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87 Figure 4-10. H/D exchange mass spectra of myoglobin in solution and on silver nanoparticles Res. 12-18 Res. 19-31 Res. 32-42 Res. 39-48Solution Solution H/D 1 min Solution H/D 4 min Ag H/D 4 min Solution Solution H/D 1 min Solution H/D 4 min Ag H/D 4 min

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88 Figure 4-11. Deuterium uptake of myoglobi n in solution and on silver nanoparticles 0 1 2 3 4 5 6 010203040506070Deuterium Uptake (Da)Exchange Time (min)Res. 12 18 Solution Ag_4 min 0 1 2 3 4 5 010203040506070Deuterium Uptake (Da)Exchange Time (min)Res. 39 48 Solution Ag_4 min 0 1 2 3 4 5 6 7 010203040506070Deuterium Uptake (Da)Exchange Time (min)Res. 32 42 Solution Ag_4 min

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89 CHAPTER 5 CONCLUSIONS AND FUTURE WORK Conclusions This thesis explored the possibility of using hydrogen/deuterium exchange mass spectrometry in the study of conforma tional change of myoglobin in solution and adsorbed on nanoparticles. Adsorption exper iments showed that the amounts of myoglobin adsorbed on different nanoparticles were significantly different, and were not directly related to the parti cle size within the range of this research. Pepsin and protease XIII were employed as the enzymes in digesting protein. Pepsin and protease XIII produced significantly different fragm entation patterns for myoglobin. Peptide fragments of myoglobin digest ed with protease XIII were smaller than those digested with pepsin. Both enzymes produced peptides which corresponded to protein coverage of over 85%. When compari ng the peptide fragments coverage, it was found that the common peptide fragments (using protease XIII digestion) for myoglob in in solution and on silver nanoparticles cover about 75% of the protein chain, implyi ng that the study of myoglobin conformational change on silver nanoparticles can be carried out using protease XIII and monitored with mass s pectrometry. The peptide fragments for myoglobin in solution and on nanoparticles are somewhat different, indicating that the adsorption on nanoparticles can affect the protein fragmentation pattern, even if the same protease is used. Filters can induce contami nation during sample preparation and care must be taken to avoid contamination and to obt ain strong mass spectrometric signals. Preliminary hydrogen/deuterium exchange results obtained using the Orbitrap mass spectrometer showed apparent differences in deuterium uptake between myoglobin in

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90 solution and on silver nanoparticles, indicating that a conformational change occurred when myoglobin is adsorbed on nanoparticles. Future Work Hydrogen back exchange into the peptide backbone needs to be inhibited and monitored by using back exchange control and by optimizing the procedures in sample preparation, transportation, and analysis. In order to obtain a better picture of the conformation change of myoglobin on nanoparticles, more systematic experiments, including more replicates and more exch ange times, are needed to elucidate the structural changes of myoglobin on nanoparticles in more detail. During the sample preparation, HPLC ma y be used to check the efficacy of the sample preparation before real sample analys is using mass spectrometry. This will also eliminate the possibility of inducing contamination dur ing sample preparation. Meanwhile, other characterization techniques, such as CD, fluorescence, DSC or NMR, may be used as complementary methods to confirm the credibilit y of the results.

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94 BIOGRAPHICAL SKETCH Yaoling Long grew up living in Guilin, Ch ina, before she became an undergraduate student studying chemistry in Peking Universi ty, Beijing, China, where she obtained her Bachelor of Science and Ma ster of Science degrees in chemistry. She started her graduate study in the Chemistry Department at the University of Florida in August 2006, supported by a financial aid fr om the graduate assistantship.