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Analysis of Lipids in Nerve Tissue by MALDI Tandem Mass Spectrometric Imaging

Permanent Link: http://ufdc.ufl.edu/UFE0024156/00001

Material Information

Title: Analysis of Lipids in Nerve Tissue by MALDI Tandem Mass Spectrometric Imaging
Physical Description: 1 online resource (135 p.)
Language: english
Creator: Landgraf, Rachelle
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The direct analysis of intact tissue samples using a laser allows the visualization of the chemical composition of that tissue in two dimensions. By knowing the distribution of compounds, one can monitor how a drug compound may distribute throughout the tissue, determine how the drug may affect the tissue, or just gain a better understanding of the composition of the tissue. The knowledge obtained from such a study may aid in the diagnosis, treatment, and prevention of diseases.
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 Rachelle Landgraf.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Yost, Richard A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024156:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024156/00001

Material Information

Title: Analysis of Lipids in Nerve Tissue by MALDI Tandem Mass Spectrometric Imaging
Physical Description: 1 online resource (135 p.)
Language: english
Creator: Landgraf, Rachelle
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The direct analysis of intact tissue samples using a laser allows the visualization of the chemical composition of that tissue in two dimensions. By knowing the distribution of compounds, one can monitor how a drug compound may distribute throughout the tissue, determine how the drug may affect the tissue, or just gain a better understanding of the composition of the tissue. The knowledge obtained from such a study may aid in the diagnosis, treatment, and prevention of diseases.
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 Rachelle Landgraf.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Yost, Richard A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024156:00001


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ANALYSIS OF LIPIDS IN NERVE TISSUE BY MALDI TANDEM MASS SPECTROMETRIC IMAGING By RACHELLE RENEE LANDGRAF A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Rachelle Landgraf 2

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To my Mom and Dad 3

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ACKNOWLEDGMENTS The Lord is my strength and my shield; my heart trusts in him, and I am helped. My heart leaps for joy and I will give thanks to him in song (or dissertation). Psalm 28:7 I must first thank God, my all in all. His love and faithfulness have made me who I am today, and my prayer is to always follow in His ways. I thank my family, Dad, Mom, Chrissy, David, Nicky, Andy, Mimi, and Amie, (and all of those who are in my life because of them) fo r always believing in me. Their prayers and guidance helped set my feet on a firm foundation, and I am eternally grateful for all of their love and support. I need to thank my advisor, Rick, for allowing my stint as a graduate student to be a time of discovery. His teaching has led my mind in sc ientific and life exploration and furthered my desire to seek the unknown. I must also thank the members of the Yost group, both past and present, for their creative insight, words of enc ouragement, methods of motivation, and inspiring conversations. I have to thank my church family, specifi cally Kristen, Kathy, and Megan, for their unending prayers and for challenging me in my daily walk. I give special thanks to Cindy and Jeff for opening their home and hearts to me in the transient stage of my graduate career. I also thank Mari, Ed, and Ge orge at Thermo (San Jose, CA) for allowing me to conduct research at their facility and their continual suppor t and assistance with instrument issues. Finally, I must thank my husband, Jeffery. Hi s love and humor have made difficult times bearable, mundane times lively, and joyous times ex traordinary. But most importantly, I want to thank him for blessing my life with the opportunity to practice the love of Ch rist on a daily basis. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAPTER 1 INTRODUCTION................................................................................................................. .13 Research Motivation............................................................................................................ ...13 Lipids......................................................................................................................................14 Traditional Mass Spectrometric Analyses.......................................................................14 Lipid Structure.................................................................................................................16 Mass Spectrometric Imaging..................................................................................................20 Definition.........................................................................................................................20 MSI Ionization.................................................................................................................22 MSI Mass Analysis..........................................................................................................24 MALDI............................................................................................................................25 Background..............................................................................................................25 Theory......................................................................................................................26 Ion Trap Mass Spectrometry...........................................................................................30 Background..............................................................................................................30 Theory and operation of 3D and 2D ion traps..........................................................33 2D linear ion trap description...................................................................................39 Theory and operation of an orbitrap.........................................................................41 Study Overview................................................................................................................. .....42 2 MALDI-LINEAR ION TRAP MICROPROBE MS/MS STUDIES OF THE EFFECTS OF DICHLOROACETATE ON LIPI D CONTENT OF NERVE TISSUE...........................43 Introduction................................................................................................................... ..........43 Experimental................................................................................................................... ........45 Instrumentation................................................................................................................ 45 Animal Treatment and Tissue Preparation......................................................................46 Tissue Analysis................................................................................................................ 47 Results and Discussion......................................................................................................... ..47 Lipid Identification..........................................................................................................4 7 Tissue Comparison..........................................................................................................52 Conclusions.............................................................................................................................58 5

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3 QUANTITATION OF LIPIDS IN NERVE TISSUE USING MALDI MASS SPECTROMETRIC IMAGING.............................................................................................59 Introduction................................................................................................................... ..........59 Experimental................................................................................................................... ........60 Animal Treatment and Tissue Preparation......................................................................60 Internal Standard and Matrix Application.......................................................................61 Instrumentation................................................................................................................ 63 Tissue Analysis................................................................................................................ 63 Results and Discussion......................................................................................................... ..64 Signal Variability............................................................................................................. 64 Internal Standard Evaluation...........................................................................................66 Quantitation.....................................................................................................................69 Tissue Comparison..........................................................................................................77 Conclusions.............................................................................................................................80 4 IMAGING OF LIPIDS IN SPINAL CO RD USING INTERMEDIATE PRESSURE MALDI-LIT/ORBITRAP MS................................................................................................82 Introduction................................................................................................................... ..........82 Experimental................................................................................................................... ........83 Animal Treatment and Tissue Preparation......................................................................83 Matrix Application..........................................................................................................84 Instrumentation................................................................................................................ 84 Tissue Analysis................................................................................................................ 86 Results and Discussion......................................................................................................... ..86 Orbitrap vs. Linear Ion Trap............................................................................................86 Imaging using High Resolution MS Scanning................................................................90 Role of HRMS and MS/HRMS in Identifying Lipids.....................................................93 Conclusions.............................................................................................................................98 5 IDENTIFICATION OF LIPIDS IS NERVE TISSUE: A COMPARISON OF IONIZATION METHODS USIN G A HYBRID LTQ/ORBITRAP....................................100 Introduction................................................................................................................... ........100 Experimental................................................................................................................... ......101 Animal Treatment..........................................................................................................101 Tissue Preparation.........................................................................................................102 Intact tissue.............................................................................................................102 Tissue extracts........................................................................................................102 Instrumentation..............................................................................................................10 2 Sample Analysis............................................................................................................103 MALDI-LTQ/Orbitrap...........................................................................................103 Nanospray-LTQ/Orbitrap.......................................................................................104 LC-ESI-MS/MS.....................................................................................................104 6

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Results and Discussion......................................................................................................... 104 LC-ESI and Nanospray..................................................................................................104 MALDI..........................................................................................................................108 Conclusions...........................................................................................................................116 6 CONCLUSIONS AND FUTURE WORK...........................................................................118 LIST OF REFERENCES.............................................................................................................123 BIOGRAPHICAL SKETCH.......................................................................................................135 7

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LIST OF TABLES Table page 2-1. Identification of eight abunda nt lipid ions present in spin al cord and sciatic nerve by MS/MS and MS3, showing the major neutral losses (NLs) observed and the lipid assignment based on MS2 and MS3 data..................................................................................48 2-2. Factor decrease of ion Inte nsity of DCA-administered tissue compared to control tissue.....56 3-1. Literature and MS data co mparison of relative concentration of phosphatidylcholines of varying fatty acid tail composition..........................................................................................72 3-2. Comparison of relative concentration (SE) of phosphatidylcholines of varying fatty acid composition in spinal cord from thr ee control (C) and three DCA-administerd animals........................................................................................................................ .............79 5-1. Mass peak assignments for lipids detected using direct infusi on nanospray (nESI) of lipid extracts, MALDI of lipid extracts (M ALDI-e), and direct analysis of tissue by MALDI (MALDI-t)...............................................................................................................109 8

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LIST OF FIGURES Figure page 1-1. Site of action of dichloracetate.......................................................................................... .....15 1-2. Structure of glycerophospholipids (GPLs).............................................................................18 1-3. Structure of sphingolipids (SPLs)......................................................................................... .19 1-4. Process of creating a mass spectrometric image....................................................................21 1-5. Common matrixes used in MALDI and their abbreviations..................................................27 1-6. Ideal 3-dimensional quadrupol e ion trap and dimensions r0 and z0.......................................32 1-7. Two different designs of the 2-dimensional quadrupole ion trap...........................................34 1-8. Orbitrap mass spectrometer................................................................................................ ....35 1-9. Small portion of the Mathieu stability diagram......................................................................37 1-10. Voltages applied to the 2D ion trap for axial trapping, radial trapping, and radial excitation..................................................................................................................... .......40 2-1. MS2 spectrum of the m/z 850.3 ion from control rat spinal cord and corresponding neutral losses; MS3 spectrum of m/z transition 850.3 763.3.........................................51 2-2. Mass spectra of the lipid region averaged ove r a section of sciatic nerve collected from control and DCA-treated rats.............................................................................................53 2-3. Ion intensity of eight abundant lipid ions found in spinal co rd tissue of one control (C7) and three DCA-treated (DCA1, DCA4, DCA5) ra ts and sciatic nerv e tissue of three control (C4, C5, C9) and three DCA-tr eated (DCA1, DCA4, DCA5) animals.................54 2-4. Mass spectra of lipid standard PC(10:0,10: 0) with no addition of DCA, addition of 10ppm DCA, and additi on of 80-ppm DCA..........................................................................57 3-1. Three approaches to applying internal standard to tissue.......................................................62 3-2. Signal intensity of four ions over the course of 20 analyses from one section of spinal cord....................................................................................................................................65 3-3. Mass spectra of the phospholip id region of serial rat brai n sections with no internal standard applied and with internal standard.......................................................................67 3-4. Mass spectral images of the [M+Na]+ ion of the internal standard, m/z 588, applied in a 3-mm square to serial sections of rat brain on top of dry tissue, on top of wet tissue, and under wet tissue........................................................................................................... 70 9

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3-5. Mass spectral images of the [M+Na]+ ion of PC(16:0,16:0), m/z 756, divided by the intensity of the internal standard, m/z 588, applied on top of dry tissue, on top of wet tissue, and under wet tissue................................................................................................71 3-6. Mass spectra of the phospholip id region averaged over the entire section of rat brain and rat spinal cord............................................................................................................ ..74 3-7. MS/MS spectrum of m/z 856 showing the ne utral losses (NL) of 43, 59, and 87, which indicates the presence of isobaric lipids.............................................................................76 3-8. Mass spectrum of the phospholipid range fro m spinal cord taken at 100,000 resolution......78 4-1. Hybrid linear ion trap/orbitrap with interm ediate pressure MALDI source adapted from MALDI LTQ Orbitrap product specificati ons from Thermo Fisher Scientific.................85 4-2. Mass spectra of the phosphlipid m/z region of a section of rat spinal cord...........................87 4-3. Mass spectrometric images of the total ion current of serial rat spinal cord sections analyzed by an orbitrap and a linear ion trap.....................................................................89 4-4. Mass spectra of phospholipids found in whit e matter and gray matter analyzed on an orbirtrap and a linear ion trap.............................................................................................91 4-5. Mass spectrometric images of m/z 756 and m/z 776 generated from serial rat spinal cord sections analyzed on an orbitrap and a linear ion trap...............................................92 4-6. Mass spectrum of m/z region 844-845 show ing at least 5 peaks detected and the corresponding mass spectrometric images.........................................................................94 4-7. Mass spectrum of m/z region 848-849 acqui red on an orbitrap and corresponding mass spectrometric images; MS/MS spectru m of m/z 848 and corresponding mass spectrometric images.........................................................................................................96 5-1. Lipid extract by LC-ESI-MS/MS.........................................................................................106 5-2. Mass spectrum of lipids detected from tissue extract by direct infusion nanospray ionization..........................................................................................................................107 5-3. Mass spectrum of lipids detected directly from tissue by MALDI......................................112 5-4. Mass spectrum of lipids detected from tissue extract by MALDI........................................115 10

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ANALYSIS OF LIPIDS IN NERVE TISSUE BY MALDI TANDEM MASS SPECTROMETRIC IMAGING By Rachelle Renee Landgraf May 2009 Chair: Richard A. Yost Major: Chemistry Direct analysis of tissue from both the centr al and peripheral nervous systems of control rats and rats administered the potential neurotoxin di chloroacetate (DCA) was investigated using an intermediate-pressure matrix-assisted laser desorption/ionization (IP-M ALDI) source coupled to a linear ion trap (LIT) mass spectromete r. The MALDI matrix, 2,5-dihydroxybenzoic acid, was applied to the tissue using a novel automated inkjet printer system. The MS/MS capabilities of the LIT allowed identification of lipids deso rbed directly from tissue. In some cases, a marked decrease is observed in the intensity of lip id ions in spinal cord and sciatic nerve from rats exposed to DCA. The results also demons trate the rapid, sensitive and semi-quantitative capabilities of this method. Further quantitative cap abilities of MALDI were examined through the use of an internal standard. A non-endogenous phospholipid, PC(10: 0,10:0), was chosen because it does not interfere with ions typically found in the li pid mass range (m/z 700 900) as well as having similar ionization and extraction e fficiency to the endogenous lipids of interest. Three different application methods of the intern al standard were evaluated; re sults show that, for quantitative purposes, application on top of dr y tissue is the most desirable method. Utilizing an internal 11

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standard allows the calculation of the rela tive concentration of endogenous lipids with a precision of better than 5% standard error. A hybrid linear ion trap/orbitrap mass spectrometer was used to perform MS/MS experiments and high resolution mass analysis of lip ids desorbed from nerve tissue. A dramatic improvement in mass spectral resolution and a decrease in background are observed in the spectra collected from the orbitrap, which allows generation of more accurate mass spectrometric images of the distribution of lipids within ne rve tissue. Employment of both mass analyzers provides a rapid and reliable means of compoun d identification based on MS/MS fragmentation and HRMS accurate mass. Finally, three different ionizat ion methods were approached fo r the analysis of lipids in nerve tissue on the hybrid linear ion trap/orbitrap mass spectrome ter. Extracted lipids were analyzed by both direct infusion nanospray and MALDI, and direct interrogation of intact tissue was performed using MALDI. Nanospray experi ments produced protonated ions that were identified as PCs, PEs, Cers, and SPMs. MALDI of intact tissue with additional Na resulted in the formation of [M+H], [M+Na], and [M+K] ions. Molecular species were identified as Cers, SPMS, PCs, PEs, and PSs. Lipid extracts anal yzed by MALDI resembled a combination of the data obtained in the other two experiments. A comparison of the three ionization techniques used here also demonstrated that lipid ions tend to more fragile when produce by MALDI compared to nanospray. The direct analysis of tissue proved to be more sensitive than the tissue extract experiments, and also affords the opportu nity to provide localiz ation of lipids within tissue 12

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CHAPTER 1 INTRODUCTION Research Motivation Congenital lactic acidosis (CLA) involves a mu ltitude of inborn errors that affects the ability of mitochondria to efficiently convert substrate fuels into adenos ine triphosphate. CLA often arises from loss-of-function mutations in genes coding for respir atory chain and other mitochondrial enzymes as well as point mu tations and deletions in mitochondrial DNA.1-4 These errors most often resu lt in an accumulation of lact ate and hydrogen ions in blood, urine, and cerebrospinal fluid. Highly oxidative tissues, such as the nervous system, skeletal muscle, heart, and kidney cortex, are particularly vulnerable to th is perturbed state, and CLA is typically associated with progr essive neurological and neuromuscular deterioration and early death. The investigational drug dichlo roacetate (DCA) has been used in the chronic treatment of CLA because of its lactate-lowe ring capabilities and potential to increase cellular energetics.5-7 Such treatment stems from the early application of the diisop ropylammonioum salt of DCA for the therapeutic treatment of human diabetes mellitus.8 Non-therapeutic human exposure has also been reported for DCA in drinking water at non-toxic levels where DC A is generated as a disinfection byproduct during chlorination.9 The lactate lowering effect of DCA is based on its inte raction with the pyruvate dehydrogenase complex (PDC), which is located in the mitochondria. PDC catalyzes the ratelimiting step in the aerobic oxidation of glucose, pyruvate, and lactate. Th e regulation of PDC is in part controlled by reversib le phosphorylation. DCA inhibits pyruvate dehydrogenase kinase involved in phosphorylation and locks PDC in its unphosphorylated, active form, which 13

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accelerates the aerobic oxidation of glucose, pyruv ate, and lactate to acetyl coenzyme A (Figure 1-1).10 However, the use of DCA has been mitigated in some patients due to reversible peripheral neuropathy,6,7 which has also been demonstrated in dosed animals at exposure levels 50 mg/kg/day for several weeks or months.11 Initial hypotheses stated that DCA-associated neuropathy could be due in part to demyelinat ion in both the central and peripheral nervous systems. Demyelination is the deterioration of the myelin sheath, a structure predominately composed of lipids (70-85% by weight).3 Therefore, it is the goal of this dissertation to characterize the lipid content of nerve tissue us ing the innovative technique mass spectrometric imaging. Lipids Traditional Mass Spectrometric Analyses Lipids play a critical role in the structure and function of th e nervous system and contribute to the specialized properties of nervous system structures such as myelin. Lipids are involved in signaling pathways, which regulate cell different iation and synaptic tr ansmission as well as modifications of proteins for receptor modulation.12 Mass spectrometry has been an integral tool for the structur al elucidation of lipids for many years. Initial studies employed electron ioni zation (EI) as the major source for generating lipid ions. Because of the low vol atility of many lipid classes, extensive derivatization measures were used for EI analysis.13 Due to the highly energetic processe s involved in EI, a great deal of fragmentation ensues during ioni zation often rendering a very w eak or nonexistent molecular ion signal. It wasnt until the 1960s, with the deve lopment of chemical ionization (CI), that lipid analysis was enhanced by the ability to de pendably obtain molecular weight information.13 14

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Figure 1-1. Site of action of DCA (a dapted from Stacpoole, P.W. et al Controlled clin ical trail of dichloroacetate for treatment of congenital lactic acidosis in children. Pe diatrics 117, 1519-1531 (2006).) A subunit of the pyruvate dehydrogenase (PDH) component of the PDC is reversibly phosphorylated and inactivated by PDH kinase. DCA inhibits the kinase, maintaining PDH in its unphosphorylated, active form. 15

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A revolution in lipid analysis came with th e invention of fast atom bombardment (FAB) in 1981.14 FAB provided a means of soft ionizati on of non-volatile compounds without the need for time-consuming derivatization processes. The matrix used in FAB allowed the analysis of intact analytes along with refreshment of the sample surface which helped lower sensitivity through accumulation of signal. Pr oducing intact molecular ions also made possible the coupling of FAB to tandem MS where identification c ould be made using collisionally induced dissociation. Another significant advancement in lipid analysis came with the introduction of electrospray ionization (ESI).15 Employing ESI, lipid ions coul d be generated directly from solution, which allowed the straightforward coup ling to liquid chromatography. By utilizing these combined techniques, another stage of anal ysis presented additional information useful for the characterization and identification of lipids. Lipid Structure The majority of lipids in nerve tissue are comple x with amphiphilic structure. They consist of both a small polar, or hydr ophilic, and a large non-polar, or hydrophobic, component. In nerve tissue, the hydrophobic component consists of a fatty acid moiety, which is a long-chain carboxylic acid that may be saturated or may contain one or more double bonds, all in the cis configuration. When multiple double bonds are present, they are nonconjugated and almost always three carbons apart. The shorthand notat ion for fatty acids consists of the number of carbon atoms followed by the number of double bonds. For example, oleic acid has 18 carbons and one double bond and would be denoted as 18:1. The hydrophilic component allows identificatio n of lipids into classes; however, the category of the lipid must first be establishe d. Lipid categories are determined based on the fundamental structure of the compound, most commonly called the backbone. This dissertation 16

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focuses on the lipid categories known as glycero phospholipids (GPLs) and sphingolipids (SPLs). The backbone of GPLs is a glycerol molecule substituted at the sn -3 carbon by a phosphate functional group. The sn notation refers to stereochemical numbering. Fatty acids previously discussed are esterified at the sn -1 and sn -2 carbons. The hydrophilic component, or head group, is ester-linked to the phosphate. The three major classes of GPLs studied here are phosphatidylethanolamines (PE), phosphatidylcho lines (PC), and phospha tidylserines (PS). Structures of all components of GPLs are shown in Figure 1-2. Sphingolipids most often cont ain a sphingosine base as the lipid backbone. They have one variable fatty acid that is acylated to the amino at the sn -2 carbon. The two classes of SPLs examined in this dissertation are sphingomyelin s (SPM), and cerebroside s (Cer). Figure 1-3 shows the structures comprising SPLs. The shorthand nomenclature of lipids througho ut this work uses the abbreviated name of the lipid class followed by the number of carbons and double bonds in the fatty acid moiety. GPLs may be identified as the total number of carbons and double bonds, or the assignment of fatty acids to the sn -1 and sn-2 positions may be given. For example, a GPL consisting of a choline head group, palmitic acid ( 16:0) and oleic acid (18:1) may be written as PC(34:1) or PC(16:0,18:1), where palmitic acid is at the sn -1 carbon and oleic acid is at the sn -2 carbon. Sphingomyelins are named according to the one amino-linked fatty acid at the sn -2 carbon, SPM(18:1). Cerebrosides may contain a sphingosine (18:1) or s phingonine (18:0) base, and the varying fatty acid may or may not be hydroxylated at the C-2 carbon. Such distinctions are made in the following manner, Cer(d18:1,24:0) and Ce r(d18:0,24:0h), where d denotes a fixed number of carbons and h corresponds to a hydroxylated fatty acid. 17

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O O OO P CH2CH H2C OO O OC CFAsn-1FAsn-2 Head group_ O O OO P CH2CH H2C OO O OC CFAsn-1FAsn-2 Head group_H2CCHCH2OH OH OH H2CCHCH2OH OH OH Head Groups Glycerol Backbone StructureCholine Ethanolamine SerineN CH3CH3H3C + N CH3CH3H3C + N CH3CH3H3C + N CH3CH3H3C N CH3CH3H3C + N H H H + N H H H + N H H H + N H H H N H H H + N H H H + O O N H H H + O O N H H H + O O N H H H + N H H H N H H H + O O sn -1 sn -2 sn -3 O O OO P CH2CH H2C OO O OC CFAsn-1FAsn-2 Head group_ O O OO P CH2CH H2C OO O OC CFAsn-1FAsn-2 Head group_H2CCHCH2OH OH OH H2CCHCH2OH OH OH Head Groups Glycerol Backbone StructureCholine Ethanolamine SerineN CH3CH3H3C + N CH3CH3H3C + N CH3CH3H3C + N CH3CH3H3C N CH3CH3H3C + N H H H + N H H H + N H H H + N H H H N H H H + N H H H + O O N H H H + O O N H H H + O O N H H H + N H H H N H H H + O O sn -1 sn -2 sn -3 Figure 1-2. Structure of glycerophospholipids (GPLs) GPLs consist of a glycerol backbone, a polar head group, and two fatty acid tails. Three major classes of GPLs are listed next to the head group structure. 18

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H2CCHCH2NH O HO CH CH (CH2)12CH3 Head group COFAsn-2 H2CCHCH2NH O HO CH CH (CH2)12CH3 Head group COFAsn-2 H2CCHCH2NH2OH HO CH CH (CH2)12CH3 H2CCHCH2NH2OH HO CH CH (CH2)12CH3 H2CCHCH2NH2OH HO CH2CH2(CH2)12CH3 H2CCHCH2NH2OH HO CH2CH2(CH2)12CH3 SphingosineBackbone SphingonineBackbone Structure Head GroupsSphingomyelin Cerebroside O H H H OH OH HOH H OH O H H H OH OH HOH H OH O H H H OH OH HOH H OH O H H H OH OH HOH H OH N CH3CH3H3C + O O O P N CH3CH3H3C + O O O P N CH3CH3H3C + N CH3CH3H3C + N CH3CH3H3C N CH3CH3H3C + O O O P H2CCHCH2NH O HO CH CH (CH2)12CH3 Head group COFAsn-2 H2CCHCH2NH O HO CH CH (CH2)12CH3 Head group COFAsn-2 H2CCHCH2NH2OH HO CH CH (CH2)12CH3 H2CCHCH2NH2OH HO CH CH (CH2)12CH3 H2CCHCH2NH2OH HO CH2CH2(CH2)12CH3 H2CCHCH2NH2OH HO CH2CH2(CH2)12CH3 SphingosineBackbone SphingonineBackbone Structure Head GroupsSphingomyelin Cerebroside O H H H OH OH HOH H OH O H H H OH OH HOH H OH O H H H OH OH HOH H OH O H H H OH OH HOH H OH N CH3CH3H3C + O O O P N CH3CH3H3C + O O O P N CH3CH3H3C + N CH3CH3H3C + N CH3CH3H3C N CH3CH3H3C + O O O P Figure 1-3. Structure of sphingolipids (SPLs). SPLs consist of a sphingosine or sphingonine base as the backbone, a polar head group, and one fatty acid tail. Two major classes of SPLs are listed next to the head group structure. 19

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Mass Spectrometric Imaging Definition Mass spectrometric imaging (MSI) is the direct interrogation of intact tissue in a discrete pattern so as to gain an understanding of the two-dimensional distribution of co mpounds within the tissue sample. A pictorial description of this process is shown in Figure 1-4. In general, any excised tissue, including both plant and animal, can be analyzed by MSI. Tissue is first prepared by sectioning on a cryostat into very thin sections, typically 10 20 m. Cryostat sectioning usually requires the use of an optimal cutting temperature medium to affix the tissue to the cutting sample stage but can interfere with mass spectrometric analysis. To circumvent this, deionized water is used in expe riments throughout this dissertation. After being sliced, the tissue section is placed on a sampling surface which, can be glass, coated-conductive glass, or metal and is sent to the mass spectrometer for analysis Depending on the microprobe of use, ions for mass analysis can be generated by direct ablation of the sample employing either laser desorption (LD) or secondary ion mass spectrometry (SIMS), or a matrix can be applied to the tissue which facilitates ionization when pulsed with a laser as in matrix-assisted laser desorption/ionization (MALDI). The imaging process is carried out by rast ering the mass spectrometric microprobe across the tissue section in a predefined, equal step-sized pattern. Each spot at which the microprobe is fired creates a mass spectrum which is stored alon g with its relative positi on. Once acquisition is complete, imaging software is employed in which individual ions can be extracted and displayed as a function of intensity versus relative posit ion, where changes in colo r represent changes in intensity. Spatial resolution in MSI is highly dependent on micropr obe spot diameter and raster step size. Ion beams of SIMS analysis offer na nometer spot sizes wherea s laser spot diameters are in the micrometer range. Typical imaging e xperiments utilize raster step sizes equal to the 20

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1. Section tissue and place on slide 2. Coat with MALDI matrix 3. Raster laser across tissue in mass spectrometer 720 740 760 780 800 820 840 m/z 0 10 20 30 40 50 60 70 80 90 100 782.75 756.85 810.71 798.68 753.89 772.75 832.64 723.87 769.84 826.66 751.87 836.70 820.62 792.01 743.40 721.65 4. Generate position specific mass spectrum 5. Extract m/z value to create image m/z 776 m/z 756 1. Section tissue and place on slide 1. Section tissue and place on slide 2. Coat with MALDI matrix 2. Coat with MALDI matrix 3. Raster laser across tissue in mass spectrometer 3. Raster laser across tissue in mass spectrometer 720 740 760 780 800 820 840 m/z 0 10 20 30 40 50 60 70 80 90 100 782.75 756.85 810.71 798.68 753.89 772.75 832.64 723.87 769.84 826.66 751.87 836.70 820.62 792.01 743.40 721.65 4. Generate position specific mass spectrum 720 740 760 780 800 820 840 m/z 0 10 20 30 40 50 60 70 80 90 100 782.75 756.85 810.71 798.68 753.89 772.75 832.64 723.87 769.84 826.66 751.87 836.70 820.62 792.01 743.40 721.65 720 740 760 780 800 820 840 m/z 0 10 20 30 40 50 60 70 80 90 100 782.75 756.85 810.71 798.68 753.89 772.75 832.64 723.87 769.84 826.66 751.87 836.70 820.62 792.01 743.40 721.65 4. Generate position specific mass spectrum 5. Extract m/z value to create image m/z 776 m/z 756 5. Extract m/z value to create image m/z 776 m/z 756 Figure 1-4. Process of creating a mass spectrometric image 21

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spot diameter of the microprobe. By increasing the step size to 2times or more the spot size, a profile of the tissue sample can be created which gives a lower-reso lution account of compound distribution but provides mu ch quicker analysis times. Highest resolution is achieved when a technique known as over-sampling is used. This method involves decreasing the step size to one-half of the spot size. Unless it is employed with samp le exhaustion, over-sampling can produce misleading intensity values in the ma ss spectrum caused by the combined sampling of an undisturbed area and a pr eviously interrogated one. A resolution consideration specific to MALD I is the application of matrix. The drieddroplet method where the matrix is deposited directly on the analyte in small volumes (~1l) by micropipette is the standard method in MALDI ma trix application. However, this method has been shown to cause analyte migration16 which leads to a misrep resentation of analyte distribution in a mass spectrometric image. To avoid analyte migration but maintain efficient ionization, matrix must be deposited in a manner th at allows sufficient wetting of the sample to facilitate co-crystallization of the matrix and anal yte as well efficient evaporation of the solvent to prevent analyte movement. Several techniques have been explored th at successfully apply matrix in such a way, which includes electrospray deposition,16 nebulization,17 airbrushing,18 sublimation,19 and inkjet printing.20 The inkjet printing met hod is used throughout this dissertation because it provides a quick means of reproducibly and evenly coating the tissue with matrix at a relatively low cost. MSI Ionization Direct interrogation of tissue samples by mass spectrometry initially focused on elemental analysis using a laser microprobe mass analyzer (LAMMA). This was primarily due to the high degree of fragmentation caused in the ionization process. Howeve r, the technique showed some success in identifying and characterizing small mo lecules including amino acids, organometallic 22

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compounds, and polymers. A limitation of the inst rument design was its requirement for thin samples through which the laser could pass, whic h often required lengt hy and tedious sample preparation procedures.21 Utilization of SIMS for direct tissue analysis allowed for less complicated sample preparation which led to an in creased collection of analyzable compounds at greater spatial resolution. The advantageous char acteristics of SIMS have led th is technique to be one of the fore-runners of current MSI analyses. SIMS doe s, however, cause extensive fragmentation of the analyte and suffers lower sensitivity due to th e analysis depth of only a few atomic layers. Because of this, nerve tissue has often been a primary candidate for SIMS analysis because of the high lipid content that does not require a large degr ee of sensitivity. Sjovall et al22 and Nygren el al23 have shown the ability of SIMS to subcellularly locali ze cholesterol and phosphocholine, the head group specific to s phingomyelins and phospha tidylcholines, in capillary blood cells utilizing a silver imprint of the cells. Other studies have shown the ability to image specific regions of the brain that represent a notable correlation to the overall anatomical structures present24,25 allowing the distinction be tween healthy and damaged structures to be evident.26 The advent of MALDI made possible the an alysis of labile and non-volatile compounds found in tissue, and is perhaps the reason this t echnique has become the most widely used for imaging purposes. While MALDI is not able to provide nanometer s pot sizes, its spatial resolution is quite acceptable, and intact molecula r ions are able to be analyzed with greater sensitivity than that of SIMS. MALDI al so offers the ability to map both small27,28 and large29-31 molecules present in tissue as well as detect site-specific drug activity.32,33 Such experiments have demonstrated that lipid content vari es in different stru ctures of the brain,34 peptides and 23

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proteins can vary in differing regions of the same structure,29 and anti-tumor drugs can localize within a tumor.35 Crecelius et al.31 have proved that imaging is no longer is limited to a twodimensional plane. Using imaging registration techniques, a mass spectra l image of a coronal mouse brain section is matched to the atlas image, and all sections are a rranged to give a threedimensional representation. MALDI imaging is th e technique used in the experiments presented in this dissertation. Most recently, a new ionization method know n as desorption electrospray ionization (DESI) has been employed in the imaging fiel d. DESI combines the soft ionization of electrospray with the direct analysis of tissu e by desorption and does so at ambient conditions with very little sample prepara tion. This technique has shown su ccess in the analysis of drugs and metabolites in tissues,36,37 anatomical distribution of lipids in brain tissue,38,39 and the forensic studies of ink samples40 and counterfeit pharmaceuticals.41 At this point in time, the spatial resolution capabilities of DESI are at be st a few hundred micrometers, which, despite is ease of operation, have limited its use to any great extent. MSI Mass Analysis Typically, mass analysis for the direct exam ination of tissue is performed on a time-offlight (TOF) mass analyzer. Char acteristics of TOF analyzers cont ributing to this are the wide range of detectable masses, good mass resolution, low ppm accuracy, and speedy analysis times.42 Tissue samples contain a wide variet y of compounds that range from only a few elements to those well over 100,000 Da, and all are capable of being detected on a TOF. The speed of TOF analyzers is of particular good use when used in conjunction with a laser microprobe because analyses can be recorded with each pulse of the laser. Identification on a TOF is performed in a number of ways incl uding post source decay, QqTOF assemblies, and TOF/TOF instruments. Tandem mass spectrometr y by post source decay is often irreproducible 24

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and substantially lengthens the analysis time. QqTOF assemblies employ a series of linear quadrupole rods where fragmentation is induced by CID, which gives highly reproducible daughter ions but limits the mass range. By coupling two TOF analyzers, a wide mass range is achieved along with consistent fragmentation by m eans of a collision cell. A drawback to this design is the incapability of detecting fragmentation of metastable ions which lowers the efficiency of this technique.42 The quadrupole ion trap (QIT) has also been used extensively in MSI experiments for the analysis of small molecules.16,18 QITs are currently limited to an m/z value of 4000 amu which does not allow the detection of large biomolecule s. However, QITs are capable of producing notably reproducible fragmentation in MSn experiments. Not only does this provide great aid in identification but reduces interferences caused by isobaric species. Reduction of interferences and mapping of fragment ions allow for a more specific mass spectrometric image to be created. The ions of interest in this dissertation fa ll in the mass range around 700 900 amu. Because of the advantages offered by QITs for small molecule detection, an ion trap is the primary mass analyzer used for the experiments outlined in the following chapters. MALDI Background Since the 1960s, analysis of surfaces by laser desorption has become an increasingly popular technique especially with the advent of MALDI, which provided a means of ionization for compounds previously rendere d outside the realm of analysis In Germany, the concept of MADLI was first investigated in 1985 by Franz Hillenkamp and Michael Karas on the influence of the laser wavelength for the desorp tion of amino acids and dipeptides.43 The technique known today was later described in 1987 by Hillenkamp and Karas through a series of experiments involving a mixture of amino acids that exhibited differing thre shold irradiances. Further 25

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experiments utilized nicotinic acid mixed in hi gh concentration with bovine albumin to produce an [M+H]+ molecular ions.44 An alternative approach applying simila r principles as the Hillenkamp and Karas experiments was established around the same tim e by Koichi Tanaka of Japan. His study included using glycerol as a so lvent and a fine coba lt powder in excess to ionize an intact molecule of lysozome.45 While todays technique typically employs a small organic acid as in the Hillenkamp and Karas investigation, Tanaka shared in the Nobel Prize in chemistry in 2002 along with John Fenn and Kurt Wthrich for their advancements in macromolecule analyses. However, both groups proposed that the matrix serv ed a 3-fold purpose that includes isolation of the analyte, energy transfer facilitating soft ionization, and ionization by chemical reactions.46 Theory The production of ions in MADLI can be br oken down into two steps, the desorption process and the ionization process. Both proced ures are extensively studied by scientists today, but the precise mechanism involved in ioniza tion is not completely understood. What is understood is the highly important role that the ma trix plays in the overall process, which makes the choice of the proper matrix crucial. A wide variety of matrixes have been employed and ionization efficiency is dependent upon the anal yte of interest. Figur e 1-5 displays several matrixes that are most commonly used. Matrix preparation first invol ves dissolving a high concentra tion of a small organic acid that absorbs at the wavelength of the laser in a solvent that is easily evaporated. A typical analyte to matrix ratio is 1:1000 for samples in solution. Analyte to matrix ratios for intact samples such as tissue are much lower and may be on the scale of 1:10 or 1:100. This dilution imbeds the analyte molecules throughout the matrix and isolates them from one another, which aids in soft desorption from the sample surface.47 26

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CN OH O OH CN OH O OH OH OH O OH OH OH O OH 2,5-dihydroxybenzoic acid DHB -cyano-4-hydroxycinnamic acid HCCA CH3O OH CH3O O OH CH3O OH CH3O O OH N O OH N O OH 3,5-dimethoxy-4-hydroxycinnamic acid Sinapinic acid or SA Nicotinic acid NA NO2NH2 NO2NH2 NH2N NH2N 3-aminoquinoline AQ para-nitroaniline PNA Figure 1-5. Common matrix es used in MALDI and their abbreviations 27

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Desorption of the matrix and analyte occurs when a sufficient energy density is achieved through short (ns) pulse durations of the laser. Successful energy deposition is dependent on the amount of energy supplied to the samp le per laser pulse (fluence, J/cm2) rather than the duration of the laser pulse (irradiance, W/cm2).48 An energy threshold must be reached for desorption to occur and is strongly matrix-dependent.49-51 Once the threshold is reached, localized sublimation of the matrix occurs along with the ablation of a portion of the surface which expands into the gas phase and carries with it intact analyte molecules. The phase transition is a s udden and explosive event and is similar to a supersonic jet expansion under vacuum conditions.52 The formation of this jet has been proven by measurements of the large initial ion veloci ties and is shown to be matrix-dependent.53 These studies are of notable interest because the velocity of the ion is directly related to the kinetic energy of the ion. Understanding such behavior could lead to more controlled ionization that would subsequently improve mass accuracy and reso lution particularly with time-of-flight mass analyzers where the measured m/z value is dire ctly related to the kinetic energy of the ion. Many theories exist as to th e actual ionization mechanism that occurs in the MALDI plume, but a consensus has been reached that supports the idea of se veral processes occurring and not just one single event. These competing pr ocesses are reflected in the mass spectrum that contains a variety of ionized species that may be protonated, depr otonated, cationized, or radicals. General acceptance of the formation of these ions is believed to be one of two categories: 1. primary ion formation; 2. secondary ion formation.48 Primary ion formation refers to ions produ ced from the neutral molecules of the sample surface and are typically considered to be mostly a contribution form the matrix. Secondary ion 28

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formation refers to ions produced by mechan isms from the primary process and usually encompasses ionization of the analyte. A strong candidate for the primary ionization step, and perh aps the most straightforward, is the photon (h) absorption of the neutral matrix molecules (M) which lead to subsequent loss of an electron and formation of a radical (M+.) as shown in Equation 1-1. After photoionization, excited-state (M*) proton transfer may take place with a neutral matrix or analyte (A) molecule (Equations 1-2 and 1-3). M + h M* M+ + e (1-1) M* + A (M H) + (A + H)+ (1-2) M* + M (M H) + (M + H)+ (1-3) Energy pooling is another proposed ionization process for primary ion formation. This involves the excitement of two matrix molecule s that pool their energy and create a neutral matrix molecule, a radical matrix ion, and an el ectron as seen in Equation 1-4. This mechanism is quite plausible because of the strong inter actions known to occur between closely packed chromophores in solids. Clusters are also know n to be produced in the MALDI plume which can ionize analyte molecules as well (Equation 1-5). MM + 2 h M*M* M + M+ + e (1-4) M*M* + A MM + A+ + e (1-5) Other possible mechanisms have been pr oposed that could produce ions but are less extensively investigated. They include disproportionate reactions for the formation of negative ions, desorption of preformed ions, thermal ionization, and pressure pulses. Secondary ionization events take place thr ough ion-molecule reacti ons in the expanding MALDI plume and may be simple or may involve multiple steps. The ions observed in the 29

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recorded mass spectrum can differ significantly fr om those produced in the primary event. Reactions that have received considerable attention are prot on-transfer, cationization, and electron-transfer reactions. A radical matrix ion produced from the primary ionization event can react with neutral matrix molecules to result in a protonated matr ix molecule and a deprot onated radical (Equation 1-6). The radical may capture a free elec tron to form a deprotonated negative ion. M+ + M (M + H)+ + (M H) (1-6) Proton transfer reactions between matrix ions and analyte molecule s result in prot onated analyte ions (Equation 1-7). This reaction is thermodyna mically favorable when proton affinities for the analyte molecules are greater than the proton a ffinity of the matrix. Formation of negative analyte ions can be achieved by extracting a pr oton from a neutral analyte molecule by a deprotonated matrix ion as seen in Equation 1-8. Such reactions can be achieved if the gas-phase b asicity of the matrix ion is higher than that of the analyt e molecule. (M + H)+ + A M + (A + H)+ (1-7) (M H) + A M + (A H) (1-8) Cationization by the addition of an alkali meta l is another process that occurs during the secondary ionization event. Cation affinities fo r analyte molecules are generally substantially smaller than proton affinities, but cationization may be promoted by adding an excess of an alkali salt to the matrix solution. Ion Trap Mass Spectrometry Background The ability to store and mass-selectively detect ions has a history that dates back several decades and has been suggested to exist in three separate ages: mass-selective detection, massselective storage and mass-selective ejection.54 In the first age, the physicists Wolfgang Paul and 30

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Helmut Steinwedel introduced a method for tr apping a range of ion masses in a quadrupole ion trap and detecting them while st ored. A number of scientists contributed to the work in the second age, where ions of a single mass were stored in the ion trap and then ejected for detection by an external electron multiplier. The third age introduced the ion trap as a storage device for a broad range of masses with the ability to sequentia lly eject ions in order of mass to be detected externally. Operation in this manner along with the addition of helium as a damping gas and alterations of the trap dimensions, led to the co mmercialized 3D ion trap (Figure 1-6) used today (Thermo Fisher Scientific). A wide variety of ionization sources are able to be coupled to the 3D ion trap, making the analysis of any ionizable specie possible. However, the ion trap does suffer a few drawbacks. The working mass range on todays analyzers ha s a high mass cut-off at m/z 4000; therefore, without the ability to produce multiply charged ions, the range of analysis is limited to molecules that fall in this mass range. An issue unique to i on traps is that of space ch arging. If the trap is filled with too many ions, a shielding effect is in duced that causes ions to be unequally affected by the quadrupolar field. This can lead to peak broadening, mass shifts, and the appearance of ghost peaks. Automatic gain control was empl oyed as a way to eliminate space charging by allowing the software to determine the length of ion injection based on a pre-scan. Lack of sensitivity is another issue faced when using a 3D ion trap based on low ion injection efficiency and loss of half the ions during ejection where io ns exit both end-caps but are only detected at one. To circumvent some of these issues, the linear ion trap, or 2D ion trap, was introduced in 2002 as two dramatically different designs. Th e first design resembled that of a triple quadrupole mass spectrometer with the sec ond quadrupole used as an ion trap.55 This design 31

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Figure 1-6. Ideal 3-dimensional quad rupole ion trap and dimensions r0 and z0 (adapted from March, R.E. Quadrupole ion trap mass spectrome try: a view at the turn of the century. International Journal of Mass Spectrometry 200, 285-312 (2000).) 32

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offered a larger space charge capacity, but ions ar e ejected axially which still causes loss of some of the ions. The second design di vided the trap into three segments with a long center section and a front and back section of equal but shorter length.56 Two slits are machined into the center section for radial ejection of i ons and are detected simultaneousl y. Both designs (Figure 1-7) offer greater space-charge capacity and increased sensitivity due to more efficient ion injection, but the second design gives better sensitivity due to ra dial ejection and dual detection of the ions. Another type of ion trap introduced commercially by Thermo Fisher Scientific in 2005 is the orbitrap (Figure 1-8). It wa s first described by Alexander Maka rov to orbitally trap ions with an electrostatic field.57 The shape of the orbitrap was m odeled after the Kingdon trap developed in the early 1920s that consisted of a wire st retched along the axis of an outer cylinder. Modifications replaced the wire with a central spindle-like electrode and the cylinder with an outer barrel-lik e electrode. Major advantages offered by the orbitrap include the following: 1. high mass resolution (up to 100,000) without the need of cryogens as in Foureir tran sform ion cyclotron resonance mass spectrometry; 2. high mass accuracy (sub pp m); 3. large space-charge capacity; 4. large dynamic range.42 The orbitrap only offers one stage of mass analysis, but recent commercialized instruments couple the orbitrap with a linear ion trap capable of performing MSn. This hybrid instrument also allows a more compatible c onfiguration for the use of continuous ionization sources with the pulsed operation of the orbitrap.58 Theory and operation of 3D and 2D ion traps The 3D QIT consists of three hyperbolic elec trodes, two end-cap electrodes and one ring electrode. The ring electrodes are virtually iden tical with each containing a small hole in the center through which ions are allowed to enter and exit. The ring electrode with radius ro is 33

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Exit Slit Exit Slit A B Figure 1-7. Two different designs of the 2-dimensional quadrupole ion trap. A) The design introduced by Thermo Fisher Scientific (adapted from Schwartz, J.C. et al A twodimensional quadrupole ion trap mass spectro meter. Journal of the American Society for Mass Spectrometry 13, 659-669 (2002).) cons ists of 3 sections. The front and back sections are shorter than the middl e section where ions are stored, ejected radially for detection, or ejec ted axially for further stages of analysis. B) The design introduced by MDS Sciex (adapted from Hager, J.W. A new linear ion trap mass spectrometer. Rapid Communications in Mass Spectrometry 16, 512-526 (2002).) consists of 3 sets of quadrupole rods of equa l length. q2 is used as a collision cell and ions are ejected axially for detection. 34

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Figure 1-8. Orbitrap mass spectrometer (adapted from Makarov, A. Electrostatic axially harmonic orbital trapping: A high-perfomance technique of mass analysis. Analytical Chemistry 72, 1156-1162 (2000).) Curved arro ws show an example of a stable ion trajectory. R1 and R2 are the maximum radii of the corresponding electrodes. 35

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situated between the end-caps at a distance of zo, the distance from the center of the ring electrode to either end-cap.59 The previously stated geometry provides a pr actical working instrument, but the existence of entrance and exit holes introduces field imperf ections affecting trapping and scanning out of ions, which often lead to mass shif ts and the appearance of ghost peaks. In order to compensate for these field imperfections, the distan ce between the end-caps is stretched, zo is increased by 10.6%.60 Once ions have entered the trap, they are su ccessfully held there by a quadrupolar field which is created by applying a radi o frequency (RF) to the ring elec trode. Stability of an ion of specific m/z within the field is defined by the foll owing Mathieu equations (s tretched geometry): (1-9) (1-10) where e is the charge of the ion, U is the DC potential, V is the amplitude of the RF, m is the mass of the ion, and is the angular frequency of the appl ied RF. Solutions to Equations 1-9 and 1-10 give coordinates in a-q space that are plotted onto the Mathieu stability diagram, which is seen in Figure 1-9. If the calculated c oordinates fall into the overlapping region of az and qz, the ion is successfully trapped. The most common mode of ope ration is performed only along the qz axis. Because a DC potential is not normally applied to the electrodes, Equation 1-9 becomes equal to zero and the only effective tra pping parameters left ar e those in Equation 1-10. Effective trapping and mass analysis also i nvolves the use of a damping gas, which is generally helium (He). The presence of a damping gas slightly raises the pressure inside the ion trap to around 10-3 Torr.42 Ions are collisionally cooled by the He gas towards the center of the 36

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Figure 1-9. Small portion of the Mathieu stab ility diagram (adapted from March, R.E. Quadrupole ion trap mass spectrometry: a view at the turn of the century. International Journal of Mass Spectrome try 200, 285-312 (2000).). Trapped ions have stable trajectories th at fall within the area shown above. In normal operation along the a0 line, qz = 0.908 is the edge of stabil ity diagram. Ions with qz values greater than this are not trapped. 37

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trap where the quadrupolar field is more uniform. This allows for more efficient trapping and avoids ion loss caused by expansion. Once the io ns have been successfully trapped, they can be scanned out of the trap in order to obtain a full scan mass spectrum. A full scan mass spectrum is obtained by linearly ramping the amplitude of th e applied RF which leads to instability of the ions in the trap and subsequent ejec tion in order of in creasing m/z. A unique characteristic of i on traps is their ability to perform tandem mass spectrometry in time, MS/MS or MSn. This is accomplished by isolating a specific i on of interest, fragmenting it, and then obtaining a mass sp ectrum of those fragments. The Mathieu stability diagram contains r and z values that correspond to the secular frequency of a trapped ion. The fundamental frequency, z,0, of an ion is calculated from the z values. Because operation involves only the z-component, the r value is not used. Values for z and z,0 are calculated using Equations 1-11 and 1-12. Isolation is achieved by applying wavebands of frequencies across the end-cap electrodes that are in resona nce with the secular frequencies of unwanted ions. These frequencies excite and eject many ions simultaneously and leav e the ion of interest. (1-11) (1-12) After isolation, the ion of interest can th en be fragmented by applying an alternating current (AC) equal to the freque ncy of the ion in the trap acro ss the end-caps. When sufficient voltages are applied, the ion begins to gain kinetic energy and incr eases in motion. This increase in motion leads to highly energetic collisions with the He buffer gas. The collisions cause an increase in the internal energy of the ion until enough energy is gained to cause fragmentation. 38

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This procedure is called collisionally activ ated dissociation (CAD) or collision-induced dissociation (CID).61 These ions can then be scanned out in order to obtain a daughter spectrum, or MS/MS spectrum, or the process may be rep eated n times on subsequent fragment ions in order to produce MSn spectra. 2D linear ion trap description The linear ion trap is composed of four hype rbolic shaped rods cut into three axial sections. The front and back portions are 12 mm in length while the middle portion is 37 mm long. Ions are trapped axially by applying separate DC voltages to all three sections while radial trapping is accomplished by applying RF in two phases to the x and y rod pairs. A two-phase supplemental AC voltage is applied across the x rods for isolation, activ ation, and ejection of ions. The necessary voltages are depicted in Figure 1-10.56 As shown in Figure 1-7, a narrow slit 30 mm long is cut into each x rod. When the appropriate AC voltage is applied to the x rods, ions are ejected radially through these small slits and are detected on both sides by electron multipliers and conversion dynodes. As in the 3D trap, field imperfections are introduced by the existence of the slits. In order to minimize the effects of these imperfections, the x rods are exte nded 0.75 mm beyond their normal position. The detection of ions is similar to th at of the 3D trap, but offers the advantage of detecting ions that are ejected on both sides of the lin ear trap, which virtually doubles the amount of ions detectable. Other advantages offered by the 2D trap include increased ion injection efficiency, increased trapping efficiency, and a larger storage volume as well as increased dynamic range. The DC potentials applied to the rod pair s can also be manipulated in order to non-mass selectively eject ions axially allowing ions to enter other phases of analysis. The major drawback of the linear trap is its susceptibility to machining errors. While ions are confined to approximately the center1 mm of the 3D trap, ions in the 2D trap are spread out over 30 mm along the z axis. Any 39

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y z y zA DC 1 DC 1 DC 2 DC 2 DC 3 DC 3 DC 1 DC 1 DC 2 DC 2 DC 3 DC 3 RF + RF + RF RF RF + RF + RF RF B x y x y GND GND AC + AC GND GND AC + AC C x y x y Figure 1-10. Voltages applied to th e 2D ion trap. A) For axial trapping. B) For radial trapping. C) For radial excitation. 40

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imperfections in the rods can cause significant di fferences in the field th at ions experience and can lead to decreased resolution.56 Theory and operation of an orbitrap The orbitrap consists of a spindle-shap ed inner electrode and a barrel-shaped out electrode with maximum diameters of 8 mm and 20 mm, respectively. An electrostatic field is generated by applying a DC voltage to the inner electrode. Opera tion of the trap is conducted at a considerably low pressure of 10-10 Torr. Ions are injected as p ackets off-axis in the r direction and perpendicular to the z-axis. Rotational motion of the ion, wh ile not used for mass analysis, is related to the ion kinetic energy and used for ra dial trapping. Circular ion motion is described by Equation 1-13 (1-13) where r is the radius of the electr ostatic analyzer as well as th e radius of the ion trajectory, eV is the ions kinetic energy, and eE is the force experienced by the ion due to the electric field. Electrostatic attraction of the i on to the inner electrode is comp ensated by the centrifugal force arising from the initial tangential velocity. Ions are squeezed toward s the center electrode by increasing the applied voltage. In the axial direction, ions are fo rced to move away form the narrow gap between the electrodes towards the wider gap near the equator, which initiates axial oscillations. After the voltage on the inner electrode is stabilized, ion trajectories become stable spirals. The precise shaping of the electrodes create a combined quadrologrithmic electrostatic potential described by Equation 1-14 (1-14) where r and z are cylindrical coordinates, C is a constant, k is field curvature, and Rm is the characteristic radius. It is noted that the z coordinate appear s only in one term. An ion of certain 41

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mass m and charge q is accelerated along the z ax is from the force created by the electric field and can be described as a simple harmonic osci llator completely independent of the radial motion. The mass/charge ratio (m/q) is simply related to the frequenc y of ion oscillation along the z-axis (Equation 1-15). (1-15) Ion detection is therefore pe rformed by broadband image current detection followed by fast Fourier transform to convert the recorded time-domain signal into a mass spectrum.62-64 Study Overview The following chapters describe the analysis of lipids in nerv e tissue including brain, spinal cord, and peripheral ne rves. Chapter 2 describes a ra pid means to compare the lipid content of spinal cord and scia tic nerve from both control and dosed animals. Semi-quantitative measurements and lipid identification using tand em MS is also demonstrated on tissue samples weighing less than 70 g. Chapter 3 investigates the ut ility of quantifying endogenous lipids using an internal standard. Si gnal variability and di ffering approaches to internal standard application are evaluated to de termine extraction and ionization efficiency of the internal standard both of which are critical for accurate quantitative measures. Chapter 4 examines the use of a hybrid LTQ/orbitrap mass spectrometer for the interrogation of nerve tissue. The high resolving power of the orbitrap is utilized fo r identification purposes and generation of highly specific mass spectrometric images. Chapter 5 compares differing ionization methods of lipids using a hybrid LTQ/orbitrap. Direct infusion and MALDI of lipid extracts as well as MALDI of intact tissue is studied for the identification of a broad range of lipid ions. Chapter 6 offers a conclusion to the areas previously discussed and a projection as to wher e tissue analysis using mass spectrometric imaging techniques may be headed in the future. 42

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CHAPTER 2 MALDI-LINEAR ION TRAP MICROPROBE MS/MS STUDIES OF THE EFFECTS OF DICHLOROACETATE ON LIPID CONTENT OF NERVE TISSUE Introduction The direct analysis of tissue sections by microprobe mass spectrometry provides an opportunity to identify changes in the spatial distribution of molecu lar species present within the tissue and the possibility of correlating these data to those obtained using histological techniques.65 When employed in conjunction with tandem mass spectrometry,27 superior specificity and structural eluc idation can be achieve d through the reduction of matrix background interferences and isolation of isobaric species. Spatial resolu tion and background reduction are of great importance when analyzing complex samp le matrices such as nerve tissue where it has been shown that the lipid content of wh ite and gray matter differ significantly.34 Although the interest of microprobe mass spectrometry is currently focused on imaging specific compounds in tissue,29 here we demonstrate the capabilities for ra pid, sensitive, and semi-quantitative analysis of tissue samples less than 70 g. Lipid composition greatly affects the functionality of neural membranes.66 Alterations in the head group, length of the fatty acid chains, an d the degree of saturation are important factors of the physicochemical properties of membrane s. Maintenance of biologically significant processes, including apoptosis and variance of activities of transpor ters and membrane-bound enzymes, is regulated by membrane lipids. In addition, lipids also se rve as reservoirs for secondary messengers. The varying developmental patterns of lipids in di ffering regions of the nervous system give rise to a diverse rati o of major lipid compone nts (sphingolipids and phospholipids).67 Marked changes in this lipid com position have been reported to occur in neurological disorders and can be manifested as changes in membrane fluidity and 43

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permeability.66 Therefore, analysis of the lipid profile of neural tissue is important to understand the mechanisms involved in neurological disorders. Of particular interest here is a neurological disorder involving the disturbance of mitochondrial function. The ability of mitochondria to efficiently convert substrate fuels into energy is a requisite for life among eukaryotic or ganisms, and interruptio n of this process can lead to several clinical complicat ions, including lactate. Congenita l forms of lactic acidosis arise from loss-of-function mutations in genes coding for respiratory chain and other mitochondrial enzymes.1-4 This results in an accumulation of la ctate and hydrogen ions in blood, urine and cerebrospinal fluid. Highly oxidative tissues, su ch as the nervous system, are particularly vulnerable to this perturbed stat e, and congenital lactic acidosis (CLA) is typically associated with progressive neurological and neuromuscular deterior ation and early death. The investigational drug dichloroacetate (DCA) has b een used in the chronic treatment of CLA, because of its lactate-lowering capabilities a nd potential to increas e cellular energetics.5-7 These effects are mediated by the interaction of DC A with the pyruvate dehydrogenase complex (PDC), which is located in the mitochondria. PDC catalyzes the rate-limiting step in the aerobic oxidation of glucose, pyruvate, and lactate. Th e regulation of PDC is in part controlled by reversible phosphorylation. DCA inhibits the kinase involved in phosphorylation and locks PDC in its unphosphorylated, active form.10 However, the use of DCA has been mitigated in some patients due to reversib le peripheral neuropathy,6,7 which has also been demonstrated in dosed animals at exposure levels 50 mg/kg/day for several weeks or months.11 We hypothesized that DCA-associated neuropathy could be due in part to a change in lipids in both the central and peripheral nervous systems where lipid cont ent can be as high as 70-85% by weight.3 44

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Traditional analytical methods used for the analysis of lipids have lacked both speed and efficiency. These conventional t echniques, which include thin-layer68 and gas69 chromatography, involve multi-step sample preparat ion procedures that are labor-intensive and time-consuming. High-performance liquid chro matography has gained popularity in lipid analysis due to the decreased need for sample preparation, but still suffers from long elution times and complicated solvent systems.70,71 In this study, we demons trate rapid lipid analysis using a novel matrix-assisted laser desorption/li near ion trap (MALDI-LIT) microprobe tandem mass spectrometer system and an automated MALD I matrix application procedure. Tandem MS capabilities (MS/MS and MSn) allow lipid identification without complicated sample preparation on samples 70 g or smaller. Experimental Instrumentation Studies were carried out on a linear ion trap fitted with a MALDI ion source that operates at intermediate pressure (Finnigan LTQ with vMALDI, Thermo Fisher Corporation, San Jose, CA), as described previously.34 Briefly, a N2 laser with a wavelength of 337 nm is directed to the source by means of a fiber optic cable. Op tics external to the vacuum chamber allow laser spot diameters in the range of 80 to 120 m that are focused at an in cident angle of 32 onto the sample plate. The source operates at a pressure of 0.17 Torr, which is ~105 times higher pressure than a traditional high-vacuum MALDI source, but ~104 times below that of an atmospheric pressure (AP) MALDI source. This intermedia te pressure reduces the amount of in-source fragmentation, compared to traditional vacuum MALDI, and yields a hi gher signal than that produced by AP MALDI. Two vacuum-rated st epper motors control the two-dimensional movement of the sample plate. A set of m odified quadrupole rods, q00, at the front of the 45

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multipole assembly permits entry of the laser beam and access of by the camera to view the sample plate. The rest of the system is id entical to that of a typical Finnigan LTQ. Animal Treatment and Tissue Preparation All animal studies were approved by the loca l IACUC at UCSD and were performed in an AAALAC-approved vivarium. Adult (250-300g body weight) female Sprague-Dawley rats (Harlan, San Diego, CA) were housed 3 per cage under a 12 hr light:dark cycle with free access to food (Harlan Teklad 7001) and water. An imals that received DCA (sodium salt, TCI America, Portland OR) were given a daily dose of 500 mg/kg by gavage for eight weeks after they achieved a weight of 250 g and were sacrifi ced one day after the fina l dose. Excised tissue was immediately flash frozen in liquid nitrogen and stored at -80C unt il shipped on dry ice in plastic tubes to the University of Florida, where it was again stored at -80 C until processed. Frozen tissue was sectioned to a thickness of 10 m at a temperature of -20C using a Leica CM1850 cryotome (Houston, TX). The tissue was att ached to the cryotome sample stage using water or a water/ice slush to avoid the ma ss spectral interferences caused by traditional techniques that use polymers to affix the tissu e to the sample stage. Tissue sections were allowed to thaw on glass microscope slides and then stored at -80 C. Before matrix application, the tissue was allowed to dry for 30 min in a vacuum dessicator. Matrix was applied to the tissue using an Epson Stylus Photo R220 inkjet printer.20 Parameters within the Epson CD printing software were adjusted to give an even coat of matrix across the entire tissue section. Evenness was monitored visually using a dissecting mi croscope with transmitted illumination through a blue filter. The microscope slides were placed in a modified CD tray holder and passed through the printer 10 times. The matrix used was 2,5-dihydroxybenzoic acid (DHB) at a concentration of 40 mg/mL in 70/30 methanol/water with an added 10 mM of sodium acetate in order to promote cationized lipids. 46

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47 Tissue Analysis Microscope slides affixed with coated tissu e were adhered to a modified MALDI sample plate using double-sided tape (3M, St. Paul, MN). The laser rast er size was set to 100 m, which equals the laser spot diameter. All experiment s were performed with automatic gain control turned off, and the signal was optimized by ma nually adjusting the laser power and the number of laser shots fired on each tissue specimen. Fu ll-scan MS experiments were typically run at a relative laser power of 30 (a rbitrary units) with 10 laser shots per step. MS2 and MS3 experiments usually required a relative laser pow er around 40 with 12 laser shots per step. Three serial sections of spinal cord from one c ontrol and three DCA-treated animals were each analyzed three times. Eight serial sections of sciatic nerve from three control and three DCAtreated animals were also each analyzed three tim es. Data were averaged over an entire tissue section with care taken to exclude signal from areas off of the tissue. Qualitative data were processed using standard Xcalibur software, and semi-quantitative results were processed using Microsoft Excel. Results and Discussion Lipid Identification Lipid ions produced by MALDI-MS are detected as singly charged ions in the region around mass-to-charge (m/z) 700 to 900.27 The ions chosen for analys is were identified using tandem mass spectrometry and the major product ions are presented in Table 2-1 (for basic lipid structure see Figures 1-2 and 1-3) Ions of m/z 753.4, 756.4, 782.4, 798.3, 810.4, 835.5 and 837.5 predominately exhibit neutral losses (NLs) of 59, 183 and 205. A NL of 59 is consistent with the loss of trimethylam ine from a phophatidylcholine (PC) or sphingomyelin (SPM) that has been cationized.27 Using the nitrogen rule, an ion can be identified as a PC or SPM based on its nominal mass. Protonation and cationizati on of odd numbered PCs and even numbered SPMs

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m/z Observed Major NLs m/z Transition Observed Major NLs Peak Assignment 753.459, 183 753.4 694.5124 [SPM(18:0)a + Na]+756.459, 183, 205 756.4 697.4124, 146, 256 [PC(16:0,16:0) + Na]+782.459, 183 782.4 723.4124, 146, 256, 282 [PC(16:0,18:1) + Na]+798.359 798.3 739.3124, 162 [PC(16:0,18:1) + K]+810.459, 183, 205 810.4 751.4124, 146 [PC(18:0,18:1) + Na]+835.559, 183 835.5 776.5124 [SPM(24:1) + Na]+837.559, 183 837.5 778.5124, 146 [SPM(24:0) + Na]+850.318, 59, 87, 162, 180, 338, 366850.3 791.4124 [PC(38:3) + K]+850.3 763.3282, 284 [PS(18:0,18:1) + Na + K]+850.3 688.418, 366 [Cer(24:0hb) + Na]+850.3 670.418, 366 [Cer(24:0h) + Na]+850.3 512.318, 162, 180 [Cer(24:0h) + Na]+850.3 484.417, 162 [Cer(24:0h) + Na]+MS/MS MSaVarying fatty acid tail: (x:y) x, number of carbons; y, number of double bonds; bh Fatty acid tail hydroxylated at the C2 carbon; SPM = sphingomyelin. PC = pho sphatidylcholine. PS = phosphatidylserine. Cer = cerebroside.Table 2-1. Identification of eight abundant lipid ions present in spinal cord and sciatic nerve by MS/MS and MS3, showing the major neutral losses (NLs) observed and the lipid assignment based on MS2 and MS3 data 3 48

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results in the detection of even m/z values fo r PCs and odd m/z values for SPMs. NLs of 183 and 205 correspond to the loss of th e phosphocholine head group without sodium and with sodium, respectively. Product ions resulting from the MS2 analysis of m/z 798.3 and 810.4 also include NLs equivalent to the losses of palmitic acid (NL 256, C16:0) and oleic acid (NL 282, C18:1) for m/z 798.3 and the losses of stearic acid (NL 284, C18:0) and oleic acid (NL 282, C18:1) for m/z 810.4. Comparison of intensities of the product ions allows for the assignment of the fatty acid tails. The more abundant product ion is assigned to the sn -1 carbon and the less abundant to the sn -2 carbon.72 While the abundance of these is ions is less than 1% of the most abundant ion, the wide dynamic range of the linear ion trap56 affords correct structural identification of these low abundance ions. MS3 was performed on each MS/MS product ion corre sponding to the NL of 59 in order to identify fatty acid composition. NLs of 124, 146, and 162 correspond to the loss of ethyl phosphate without a cation, with sodium, and with potassium, respectively. Product ions arising from the transitions m/z 756.4 697.4 and 782.4 723.4 yield MS3 product ions matching the losses of palmitic acid (NL 256, C16:0) for both m/z transitions and oleic acid (NL 282, C18:1) for m/z 723.4. MS4 was performed on the MS3 product ions identified as SPMs, however no further structural information was obtained. Th erefore, identification of these ions was made using a database developed by us that contains a list of th e molecular weights of major phospholipids with varying fatty acid composition and ionization states ([M+H]+, [M+Na]+, and [M+K]+). This classification is simplified becaus e SPMs typically have only one varying fatty acid tail. When analyzing small molecules in a complex sample such as nerve tissue, the likelihood of detecting isobaric species is extremely high. One such example can be seen by performing 49

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tandem MS on m/z 850.3 from any one of the ti ssue samples used in this study. The MS2 spectrum of this ion taken from a control section of spinal cord is illustrated in Figure 2-1A. A NL of 18 is indicative of a loss of water, which is observed for both lipid ions and matrix ions. The NLs of 59, 87, and 162 suggest the presence of a PC, a phosphatidyl serine (PS) and a cerebroside. The classification of th ese ions is described below. First, the NL of 59 (detected at m/z 791.4) is again attributed to the loss of trimethylamine, and MS3 of m/z 850.3 791.4 confirms the loss of ethyl p hosphate producing a major ion at m/z 667.4. While no ions relating to the fatty acid composition were obtained from the MS3 spectrum, an expansion in the m/z region 530-600 of the 850.3 MS2 spectrum provides ions for possible identification. The product i ons at m/z 594.6, 568.2, and 540.3 from the MS2 spectrum correspond to losses of palmitic acid (NL 256, C1 6:0), oleic acid (NL 282, C18:1), and gadoleic acid (NL 310, C20:1), respectiv ely. Possible identification could include a number of combinations of these and other fatty acids. Once more, because no fatty acid composition was obtained from the isolation of m/z 791.4, this PC can be classified in general as [PC(38:3) + K]+. The second isobaric species exhibi ts a NL of 87 (detected at m/z 763.4), which represents the loss of the serine head group (C3H5NO2) from a PS. Fatty acid assignment is readily obtained from the MS3 transition of 850.3 763.3 (Figure 2-1B). The product ions at m/z 481.3 and 479.2 are a result of the losses of oleic (C18:1) acid and stearic acid (C18:0), respectively. This ion is thus id entified as [PS(18:0, 18:1) + Na + K]+. The final species identified at m/z 850.3 produces fragment ions that correspond to NLs of 162 and 180. These NLs indicate the losses of C6H10O5 from a galactose sugar and the entire galactose unit (C6H1206) from a cerebroside. MS3 performed on the m/z transitions of both 850.3 688.4 322.3 and 850.3 670.4 304.3 exhibit a NL of 366, which also appears in the 50

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NL 338 Cer NL 366 Cer NL 180 Cer NL 162 Cer 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z 0 20 40 60 80 100Relative Abundance 484.3 688.6 763.4 832.6 512.4 791.4 568.2 451.3 594.6 540.3 x3 670.5 NL 18 NL 59 PC NL 87 PS A 250 300 350 400 450 500 550 600 650 700 750 800 m/z 0 20 40 60 80 100Relative Abundance 479.2 763.3 NL 284 NL 282 m/z 763.3 m/z 481.3 481.3 BNa + K + O P O O O O O O O NH 2 O O (H 2 C) 7 (CH 2 ) 16 CH 3 (H 2 C) 7 CH 3 m/z 479.2 NL 338 Cer NL 366 Cer NL 180 Cer NL 162 Cer 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z 0 20 40 60 80 100Relative Abundance 484.3 688.6 763.4 832.6 512.4 791.4 568.2 451.3 594.6 540.3 x3 670.5 NL 18 NL 59 PC NL 87 PS NL 338 Cer NL 366 Cer NL 180 Cer NL 162 Cer 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z 0 20 40 60 80 100Relative Abundance 484.3 688.6 763.4 832.6 512.4 791.4 568.2 451.3 594.6 540.3 x3 x3 670.5 NL 18 NL 59 PC NL 87 PS A 250 300 350 400 450 500 550 600 650 700 750 800 m/z 0 20 40 60 80 100Relative Abundance 479.2 763.3 NL 284 NL 282 m/z 763.3 m/z 481.3 481.3 BNa + K + O P O O O O O O O NH 2 O O (H 2 C) 7 (CH 2 ) 16 CH 3 (H 2 C) 7 CH 3 m/z 479.2 250 300 350 400 450 500 550 600 650 700 750 800 m/z 0 20 40 60 80 100Relative Abundance 479.2 763.3 NL 284 NL 282 m/z 763.3 m/z 763.3 m/z 481.3 m/z 481.3 481.3 BNa + K + O P O O O O O O O NH 2 O O (H 2 C) 7 (CH 2 ) 16 CH 3 (H 2 C) 7 CH 3 m/z 479.2 Figure 2-1. A) MS2 spectrum of the m/z 850.3 ion from c ontrol rat spinal cord and corresponding neutral losses. The losses indicate the presence of at least three isobaric lipids that have been identified as a phosphatidylchol ine (PC), a phosphatidylserine (PS) and a cerebroside (Cer). B) MS3 spectrum of m/z transition 850.3 763.3. The MS2 neutral loss of 87 is i ndicative of the loss of the serine head group (C3H5NO2) from a phosphatidylserine (PS). The MS3 major losses correspond to stearic acid (NL 284, C18:0) and oleic acid (NL 282, C18:1). This fragmentation allows identification of this ion as [PS(18:0,18:1) + Na + K]+ (structure shown as inset above). 51

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52 MS2 spectrum (850.3 484.3). The product ions at m/z 484.3 and 512.3 are typical fragments produced by a signifi cant number of cerebrosides73 and result from the loss of the fatty acid tail from the sphingosine base. The peak detected at m/z 512.3 in the MS2 spectrum is observed when fragmentation occurs after th e carbonyl carbon on the amide linked fatty acid, and m/z 484.4 is a result of the loss of the entire fatty acid tail. It is lik ely that the NL of 366 is attributed to the loss of hydroxyl ated lignoceric acid (C24:0h) due to the el evated abundance of hydroxylated cerebrosides in neuronal tissue.74 This ion is therefore identified as [Cer(24:0h) + Na]+. Tissue Comparison Figure 2-2 shows mass spectra (signal intensity from the first analysis of each section) averaged over the entire section of sciatic nerve from (A) contro l and (B) DCA-treated animals. Although the relative abundan ce of the lipid ions in tissue fr om both animal groups is quite similar, there is a 2.5-fold decrease in signal in tissue exposed to DCA, compared to control tissue. The abundant ions in the m/z 700-900 re gion correspond to phospho lipids, as described above. Many of the abundant ions above m/z 900 originate from DHB matrix clusters (seen both on and off tissue). Ions below m/z 700 can be attributed to DHB matrix ions and possibly lysophospholipids (intermediates involved in biosynthesis and metabo lism of phospholipids).75 A comparison of the ion intensities of eight abundant lipid ions from control and DCAexposed tissue is depicted in Fi gure 2-3. Data collected from sp inal cord tissue of one control (C7) and three rats administered DCA (DCA1, DCA4, DCA5) are shown in Figure 3A. Three 10m serial sections were cut from the spinal cord of each animal and analyzed three times at a laser power of 30 with 10 laser s hots per raster step across the entire tissue section. Due to ablation of matrix during MALDI, the signal decreased by an average factor of 1.7 between the first and second analyses and an average of 2.2 be tween the first and third analyses. Therefore,

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53Figure 2-2. Mass spectra of the lip id region averaged over a section of sciatic nerve collected from (A) control and (B) DCA-tr eated rats 810.7 837.6 850.8 850.7 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 0.32 0.64 0.96 1.28 1.60 0 0.82 1.64 2.46 3.28 4.10Counts (105) 782.6 837.7 756.7 958.4 589.5 727.0 551.2 518.6 672.6 918.7 986.4 782.5 810.6 760.5 518.5 958.2 723.5 575.0 615.3 672.4 551.0 986.4 940.6 697.4A BCounts (105)810.7 837.6 850.8 850.7 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 0.32 0.64 0.96 1.28 1.60 0 0.82 1.64 2.46 3.28 4.10Counts (105) 782.6 837.7 756.7 958.4 589.5 727.0 551.2 518.6 672.6 918.7 986.4 782.5 810.6 760.5 518.5 958.2 723.5 575.0 615.3 672.4 551.0 986.4 940.6 697.4A BCounts (105)

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0 2 4 6 8 10 12 1412345678Counts (104) C7 DCA1 DCA4 DCA5 0 2 4 6 8 10 12 14 753.4756.4782.4798.3810.4835.5837.5850.3m/zCounts (104) C4 C5 C9 DCA1 DCA4 DCA5A B 0 2 4 6 8 10 12 1412345678Counts (104) C7 DCA1 DCA4 DCA5 0 2 4 6 8 10 12 14 753.4756.4782.4798.3810.4835.5837.5850.3m/zCounts (104) C4 C5 C9 DCA1 DCA4 DCA5 0 2 4 6 8 10 12 1412345678Counts (104) C7 DCA1 DCA4 DCA5 0 2 4 6 8 10 12 14 753.4756.4782.4798.3810.4835.5837.5850.3m/zCounts (104) C4 C5 C9 DCA1 DCA4 DCA5A B Figure 2-3. Ion intensity of eight ab undant lipid ions found in A) sp inal cord tissue of one control (C7) and three DCA-treated (DCA1, DCA4, DCA5) rats and B) sciatic nerve tissue of three control (C4, C5, C9) and three DCA-treated (DCA 1, DCA4, DCA5) animals. The error bars are 1 standard error. Identification of each ion is the following (left to right): [SPM(18:0) + Na]+; [PC(16:0,16:0) + Na]+; [PC(16:0,18:1) + Na]+; [PC(16:0,18:1) + K]+; [PC(18:0,18:1) + Na]+; [SPM(24:1) + Na]+; [SPM(24:0) + Na]+; isobaric lipids. 54

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all intensities were norma lized by a factor of 1.7 for analysis 2 and a factor of 2.2 for analysis 3. Data from sciatic nerve tissu e of three control (C4, C5, C9 ) and three DCA-treated (DCA1, DCA4, DCA5) animals are shown in Figure 3B. In this case, eight serial sections of sciatic nerve from each animal were analyzed, and sign al from only the first analysis of each tissue section was used for comparison purposes. The ion signal for the major lipids is reproducible ( 20% SE) among control and DCA-exposed tissue for both spinal cord and sciatic nerve. A consistent decrease in ion si gnal intensity between control and DCA-exposed tissue is observed for both spinal cord and sciatic ne rve (Table 2-2). The average decrease in the intensity of lipids measured in spinal cord was 4.7, while the averag e decrease in the lipids measured in sciatic nerve was 6.2. Preliminary studies have shown that a maxi mum of 80-ng/mg DCA is detected in nerve tissue (unpublished results). E xperiments were conducted in wh ich a solution of 10-ppm and 80ppm DCA was mixed with a solution of 10-ppm PC (10:0,10:0) on a standard MALDI plate. The mass spectra produced from this study can be seen in Figure 2-4. Figure 4A is the mass spectrum of PC(10:0,10:0) with no DCA added, a nd Figures 2-4B and 2-4C show the results from the addition of 10-ppm and 80-ppm DCA, respectively. The ions at m/z 566 and 588 correspond to the [M+H]+ and [M+Na]+ molecular ions of PC(10:0,10:0), and the other ion signal is a contribution from the MALDI matrix. No marked decr ease in lipid ion signal is observed in the spectra. This shows that the decr ease in signal discussed pr eviously is not due to ion suppression caused by the presence of DCA. DCA administration was initially hypothesized to have a greater effect on peripheral nerves du e to the manifestation of peripheral neuropathy with prolonged dosing.12,15 However, these data show no statistical difference (tcalculated < ttable at 95% confidence) between the impact on the cen tral nervous system and peripheral nervous 55

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Table 2-2. Factor decrease of ion Intensity of DCA-administered tissue compared to control tissue m/z Peak assignmentSpinal cordSciatic nerveAverage 753.4 [SPM(18:0) + Na]+4.5 4.74.6 756.4 [PC(16:0,16:0) + Na]+5.9 5.85.9 782.4 [PC(16:0,18:1) + Na]+5.2 7.16.2 798.3 [PC(16:0,18:1) + K]+5.2 4.85.0 810.4 [PC(18:0,18:1) + Na]+4.7 6.65.7 835.5 [SPM(24:1) + Na]+4.1 7.35.7 837.5 [SPM(24:0) + Na]+4.1 7.96.0 850.3Isobaric Lipids 4.0 5.14.6 Average 4.7 6.25.5 56

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Figure 2-4. Mass spectra of lipid standard PC (10:0,10:0) with A) no addition of DCA, B) addition of 10-ppm DCA, and C) addition of 80-ppm DCA. The ions at m/z 566 and 588 correspond to the [M+H]+ and [M+Na]+ molecular ions of the lipid, respectively. 250 300 350 400 450 500 550 600 m/z 0 0.88 1.76 2.64 3.52 4.40 0 0.75 1.50 2.25 3.00 3.75 0 0.45 0.89 1.34 1.78 2.23 588.4 566.5 273.2 529.4 330.8 375.2 551.1 449.5 352.9 413.4 397.2 588.4 566.5 551.1 375.2 330.8 352.9 529.5 273.2 397.2 449.5 313.1 413.5 588.5 566.5 529.4 273.2 375.2 551.1 330.9 352.9 397.2 449.6 313.2 413.5Counts (105)A B C 250 300 350 400 450 500 550 600 m/z 0 0.88 1.76 2.64 3.52 4.40 0 0.75 1.50 2.25 3.00 3.75 0 0.45 0.89 1.34 1.78 2.23 588.4 566.5 273.2 529.4 330.8 375.2 551.1 449.5 352.9 413.4 397.2 588.4 566.5 551.1 375.2 330.8 352.9 529.5 273.2 397.2 449.5 313.1 413.5 588.5 566.5 529.4 273.2 375.2 551.1 330.9 352.9 397.2 449.6 313.2 413.5Counts (105)A B C 57

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system lipids. Further investigation of the eff ect of DCA on lipid synthesis in nerve tissue is required to explain this finding. Conclusions The results show that IP-MALDI/MSn may be used to analyze neuronal tissue for lipid identification and comparison. The data show the potential of this technique for rapid sample analysis (< 3 hr) without complicated sample pre-treatment performed on relatively small sample sizes. Tandem MS successfully identified individual ions as phosphatidylcholines and sphingomyelins and also identifi ed isobaric species as phosphatidylcholines, phosphatidylserines and cerebrosides. The sensitivity of this method has also been illustrated by the marked decrease in lipid intensities in both spinal cord a nd sciatic nerve of rats treated with DCA. 58

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CHAPTER 3 QUANTITATION OF LIPIDS IN NE RVE TISSUE USING MALDI MASS SPECTROMETRIC IMAGING Introduction MALDI mass spectrometric imaging has recently gained popularity for the in situ analysis of thin tissue sections. The robustness and relatively simple sample handling procedures have aided in promoting the widespread use of th is methodology. The technique has successfully demonstrated the ability to identify and characterize, in two dimensions, a host of compounds including both endogenous and exogenous species.76 Creating ion-specific images has become an invaluable tool for correlating histological f eatures to the absence or presence of diseased tissue,77,78 localization of pharmaceutical compounds and their metabolites,79,80 and monitoring the anatomical distribution of both large81-83 and small34,84-86 biomolecules. The growth of the technique has currently generated interest in moving MALDI imaging beyond a qualitative method into a quantitative one. Recent quantitative efforts employing MALDI (without imaging) have shown success in quantifying sulfatides in human serum87 and cyanobacterial toxins in cultures.88 A quantitative evaluation of sphingomyelins and glucosylceramides was performed by Fujiwaki and colleagues89 on tissue from patients with Niemann Pick and Gaucher disease; however, th e tissue was homogenize d and interrogated as spots on a MALDI plate. Each of these stud ies demonstrates the capabilities of making quantitative measurements with MALDI, but in each case, the spatial information obtained from the imaging process was not retained. Because of inherent signal variability, direct microprobe analysis of tissue has generally been considered a qualitative technique. This va riability is mostly caused by an inhomogeneous sample surface and inhomogeneous crystallization of the matrix. The impact of laser shot-toshot variability is minimized by using multiple laser shots (typically 10) to fill the ion trap for 59

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each mass spectrum. In addition, careful choices of matrixes, optimization of solvent systems, improved matrix deposition techniques, and the use of suitable internal standards have the potential to minimize MALDI signal variability and provide a rapid method of quantitation. In this study, signal variability in sequential tissue analyses and di ffering approaches to the application of an internal standard to nerve tissue are eval uated. The non-endogenous phosphatidylcholine, PC(10:0,10:0), was chosen to provide similar ionization and extraction efficiency to the phospholipids of interest. Potential migration a nd uniformity of application of the internal standard are monitored for each appr oach to internal standa rd application. MALDIMS imaging experiments are carried out to study the effect of applying an internal standard on the distribution of endogenous compounds. Finally, th e internal standard is used to calculate mol% of endogenous phospholipids, and the results are compared to data found in literature. Experimental Animal Treatment and Tissue Preparation All animal studies were approved by the loca l IACUC at UCSD and were performed in an AAALAC-approved vivarium. Adult (250-300g body weight) female Sprague-Dawley rats (Harlan, San Diego, CA) were housed 3 per cage under a 12 hr light:dark cycle with free access to food (Harlan Teklad 7001) and water. An imals that received DCA (sodium salt, TCI America, Portland, OR) were given a daily dose of 500 mg/kg by gavage for eight weeks after they achieved a weight of 250 g and were sacrifi ced one day after the fina l dose. Excised tissue was immediately flash frozen in liquid nitrogen a nd stored at -80C in plastic tubes until shipped on dry ice to the University of Florida, where it was again stored at -80 C until processed. Frozen tissue was sectioned to a thickness of 10-m at a temper ature of -25C using a Leica CM1850 cryotome (Houston, TX). The tissue was att ached to the cryotome sample stage using 60

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water to avoid the mass spectral interferences ca used by traditional techniques that use polymers to affix the tissue to the sample stage. Internal Standard and Matrix Application Sectioned tissue was thaw-mounted on both uncoa ted glass microscope slides and slides that were first coated with a 100-ppm solution of the internal standa rd, PC(10:0,10:0) in 70:30 methanol:water. Slides prepared for mounting of spinal cord were coated completely with the internal standard, while those prepared for mounting of brain sections were coated with one, 3x3mm square of the internal standa rd. The tissue mounted on uncoat ed slides was then separated into two sets. One set was immediately coated with a 10-ppm soluti on (70:30 methanol:water) of the internal standard while still wet. A lo wer concentration was app lied on top of tissue to avoid masking of the signal from endogenous lipid s. The other set of tissue was dried in a vacuum desiccator for 30 minutes and then coat ed with a 10-ppm solution of the internal standard. All tissue was then vacuum dried fo r 30 minutes and coated with matrix, 40 mg/ml 2,5-dihydroxybenzoic acid (DHB) with 10-mM s odium acetate in 70:30 methanol:water. A demonstration of the procedure is illustrated in Figure 3-1. Both the internal standard and the matrix solutions were applied using an Epson R260 inkjet printer.20 The microscope slides were placed in a modified CD tray hol der and passed through the printer 20 times for internal standard application and 15 times for matrix application. It was determined that a 10-ppm solution of the internal standard deposited 2.5 pg/mm2 per pass. Therefore, 20 passes of the 10-ppm solution deposited 50 pg/mm2, and 20 passes of the 100-ppm solution deposited 500 pg/mm2. Parameters within the Epson CD printing software were set for photo quality printing on glossy paper. Evenness was monitored visually using a dissecting microscope with transmitted illumination through a blue filter. 61

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Under wet tissue Apply internal standard to slide Place sectioned tissue on top of internal standard then dry in vacuum desiccator Coat with matrixExtraction of internal standard through tissueOn top of wet tissue Place sectioned tissue on top of slide Apply internal standard to tissue then dry in vacuum desiccator Coat with matrix On top of dry tissuePlace sectioned tissue on top of slide then dry in vacuum desiccator Apply internal standard to tissue then dry in vacuum desiccator Coat with matrix glass slide matrix dry tissue wet tissue internal standard Under wet tissue Apply internal standard to slide Place sectioned tissue on top of internal standard then dry in vacuum desiccator Coat with matrixExtraction of internal standard through tissueUnder wet tissue Apply internal standard to slide Place sectioned tissue on top of internal standard then dry in vacuum desiccator Coat with matrixExtraction of internal standard through tissueOn top of wet tissue Place sectioned tissue on top of slide Apply internal standard to tissue then dry in vacuum desiccator Coat with matrixOn top of wet tissue Place sectioned tissue on top of slide Apply internal standard to tissue then dry in vacuum desiccator Coat with matrix On top of dry tissuePlace sectioned tissue on top of slide then dry in vacuum desiccator Apply internal standard to tissue then dry in vacuum desiccator Coat with matrix On top of dry tissuePlace sectioned tissue on top of slide then dry in vacuum desiccator Apply internal standard to tissue then dry in vacuum desiccator Coat with matrix glass slide matrix dry tissue wet tissue internal standard glass slide matrix dry tissue wet tissue internal standard Figure 3-1. Three approaches to ap plying internal standard to tissu e. These techniques depict the internal standard being app lied to discrete areas of th e tissue and slide; however, some experiments were performed with inte rnal standard applie d everywhere on the tissue and slide. 62

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Instrumentation Studies were carried out on a linear ion trap fitted with a MALDI ion source that operates at intermediate pressure (Finnigan LTQ with vMALDI, Thermo Fisher Corporation, San Jose, CA), as described previously.27 Briefly, a N2 laser with a wavelength of 337 nm is directed to the source by means of a fiber optic cable. Op tics external to the vacuum chamber allow laser spot diameters in the range of 80 to 120 m that are focused at an in cident angle of 32 onto the sample plate. The source operates at a pressure of 0.17 Torr, which is ~105 times higher pressure than a traditional high-vacuum MALDI source, but ~104 times below that of an atmospheric pressure (AP) MALDI source. Two vacuum-rate d stepper motors control the two-dimensional movement of the sample plate. A set of m odified quadrupole rods, q00, at the front of the multipole assembly permits entry of the laser be am and access by the camera to view the sample plate. The rest of the system is identi cal to that of a typical Finnigan LTQ. Tissue Analysis Microscope slides affixed with coated tissu e were adhered to a modified MALDI sample plate using double-sided tape (3M, St. Paul, MN). The laser rast er size was set to 100 m, which equals the laser spot diameter. All experiment s were performed with automatic gain control turned off, and the signal was optimized by ma nually adjusting the laser power and the number of laser shots fired. Optimum mass spectra were based on a signal of at least 103 for the lipid base peak (m/z 782.5) with little to no mass shifting, which indicated no affects of space charging. Full scan MS experiments were typically run at a relative laser power of 30 (arbitrary units) with 10 laser shots per step. MS2 and MS3 experiments usually required a slightly higher relative laser power with the same number of laser shots per step as in full scan experiments. To monitor lateral migration of PC(10:0,10:0) within tis sue, three serial secti ons of brain tissue were treated as previously described. Two sections were placed on uncoated slides and a 3x3-mm 63

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square of internal standard was applied to one wet section and to one dry section. The third section was placed on top of a 3x3-mm square printe d onto a glass slide. Quantitative data were acquired from spinal cord taken from the cervical portion of the spine and from a saggital section of the brain. Tissue images were generated by in terrogating serial sections of the spinal cord from the lumbar region of the spine and 3-mm2 area of serial coronal brain sections. Quantitative data were averaged over an entire ti ssue section, with care taken to exclude signal from areas off of the tissue. Qualitative data we re obtained using standard Xcalibur software and ImageQuest (both from Thermo Fisher Scien tific) and Surfer imaging software (Golden Software, Golden, CO), and quantitative result s were processed using Microsoft Excel. Results and Discussion Signal Variability To generate reliable quantitativ e data, signal stability is of great importance. To counteract signal variability inherent to MALDI, typical experiments on a time-of-flight mass spectrometer average anywhere from 100 to 1000 laser shots per spot.90-93 This may also be done in an attempt to exhaust the sample; however, such anal yses are not possible w ith an ion trap in a single scan. The number of laser pulses must be optimized to fill the trap without creating a space charge effect; software limitations are set at a maximum of 20 laser shots per scan. At relatively high laser attenuati on, 10 laser shots produce desira ble spectra without space charging.18 When compared to conventional MALDI/ TOF methods, this lower number of laser shots could introduce greater variability. To reduc e this variability, each spot on the sample was interrogated multiple times. Figure 3-2 is a graph th at plots the signal intensity of four ions over the course of 20 analyses of a spinal cord secti on at a relative laser power of 30, which represents 200 laser shots at one spot The first analysis is dominate d by the DHB matrix at m/z 273, and 64

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Figure 3-2. Signal intensity of four ions over the c ourse of 20 analyses from one section of spinal cord. A relative laser power of 30 and 10 la ser shots were used for each analysis. DHB: 2,5-dihydroxybenzoic acid m/z 273 [2M + H 2H2O]+; Cholesterol: m/z 369 [M + H H2O]+; SPM: sphingomyelin m/z 753 [M + Na]+; PC: phosphatidylcholine m/z 756 [M + Na]+ 0 20000 40000 60000 80000 100000 120000 140000 1234567891011121314151617181920 Analysis NumberAbsolute Counts DHB Cholesterol SPM(18:0) PC(16:0,16:0) 0 20000 40000 60000 80000 100000 120000 140000 1234567891011121314151617181920 Analysis NumberAbsolute Counts DHB Cholesterol SPM(18:0) PC(16:0,16:0) 65

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66 while it remains a relatively in tense ion, increased signal is ob served among the lipid ions in subsequent analyses. Signal for all th e ions begins to tail off around the 11th examination (110 laser shots) and appears to be exhausted by the 20th (200 laser shots). The signal increase of DHB observed in the 15th analysis is most likely due to inhomogeneous crystallization. The ratios of the intensities of the three lipid ions in Figure 3-2 are fairly repr oducible. Nevertheless, by taking the average si gnal of the first ten or eleven analyses, a greater precision for quantitation is achieved. Internal Standard Evaluation Initial studies were designed to evaluate the utility of a non-endogenous phospholipid as an internal standard in ne rve tissue. A phospholipid was chosen as an internal standard to provide similar ionization and extraction e fficiency between the analytes a nd the internal standard. The phospholipid PC(10:0,10:0) is not known to occur naturally with in the tissue of the nervous system, therefore allowing the inte rference-free analysis of endogenous lipids. Figure 3-3A is a full-scan mass spectrum of the lipid region of a coronal brain section in the central gray substance region without the appl ication of the internal sta ndard; Figure 3-3B is the mass spectrum of the same area of an adja cent serial brain section with 50 pg/mm2 of internal standard applied on top of dry tissue. The characteristic molecular ions of lipids are observed in the m/z region 700-900. The [M+H]+, [M+Na]+ and [M+K]+ ions of the internal standard are seen at nominal masses 566, 588, and 604, respectively. Th is m/z region is relatively free from background interference; however, signal from ions at m/z 566 and 604 shows a slight interference for the [M+H]+ and [M+K]+ ions. MS/MS was performed on these ions from a blank tissue sample to confirm that they were not a contribution from PC (10:0,10:0). Therefore, the [M+Na]+ ion at m/z 588 was chosen for further analysis.

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67Figure 3-3. Mass spectra of the phos pholipid region of serial rat brain sections A) with no internal standard applied and B) with internal standard. The [M+H]+, [M+Na]+, and [M+K]+ ions of the internal st andard are labeled in B 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 782.6 798.5 810.6 826.6 756.6 835.7 958.2 850.8 582.9 566.9 723.5 782.7 810.7 798.6 835.7 588.5 850.8 760.7 604.5 566.6 958.3 723.7 544.5A B [M+Na]+[M+H]+[M+K]+ 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 782.6 798.5 810.6 826.6 756.6 835.7 958.2 850.8 582.9 566.9 723.5 782.7 810.7 798.6 835.7 588.5 850.8 760.7 604.5 566.6 958.3 723.7 544.5A B 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 782.6 798.5 810.6 826.6 756.6 835.7 958.2 850.8 582.9 566.9 723.5 782.7 810.7 798.6 835.7 588.5 850.8 760.7 604.5 566.6 958.3 723.7 544.5A B [M+Na]+[M+H]+[M+K]+

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Also noticeable in the two spectra is a cha nge in relative ion intensity among the lipids, most notably m/z 798 [PC(16:0,18) + K]+ and 810 [PC(18:0,18:1) + Na]+. These two ions are adducted with different cations and have been shown to be distributed differently throughout tissue.18 The sodiated lipid at m/z 810 is pred ominantly found in white matter whereas the potassiated lipid at m/z 798 is pr imarily found in gray matter, whic h may suggest a difference of ionization efficiency between white and gray ma tter once an internal standard is applied. However, further examination shows that other potassiated species exhibit a decrease in ion intensity as well. It has been reported that the sodium and potassium levels in gray matter of the spinal cord are slightly higher than in the white matter, but the rati o of the cations is the same in each.94 Intensities of the sodiated PC(10:0,10:0) ion at m/z 588 extracted from white and gray matter separately show no signifi cant difference, which implies si milar extraction and ionization efficiencies. Therefore, the change seen between the spectra can be accounted for as additional sodium ions being applied with the internal standard. A primary concern in using a phospholipid as an internal standard is the potential lateral migration of the lipid within the tissue. To monitor possible migration, an inkjet printer was used to apply a 3-mm (13%) square of internal standard under and on top of tissue, as described in the Methods section. The mass spectral images of m/z 588 extracted from tissue with internal standard applied squares on top of wet tissue, on top of dry tissu e, and under wet tissue can be seen in Figure 3-4. Previous research in our lab95 has generally shown that migration does not exceed the diameter of the laser spot size (100 m); here, no evidence of migration greater then the error in inkjet applicati on is seen. The most intense and even signal is produced from applying internal standard to the top of dry tissue. A slightly mo re intense signal is observed in the image where internal standard was applied un der wet tissue when comp ared to application on 68

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69 top of wet tissue. This demonstrates that the in ternal standard is effi ciently extracted from under the tissue and suggests possible extraction in th e opposite direction when applied on top of wet tissue. An anomaly in signal is noticed in the bo ttom left of each image in Figures 3-4A and 34B, where internal standard was applied on top of tissue. Because these samples had internal standard applied at the same time and the abnormal data are seen in the same area, it is likely that a printer error occurred and caused an uneven coat of internal standard to be applied. A slight unevenness is also observed from the top to the bottom of the image in Figure 3-4C. The effect of applying an internal standard on the distribution of endogenous lipids was also investigated by imaging mass spectrometry. The entire area of a ti ssue section including a small margin around the tissue was interrogated to cr eate an image representative of actual tissue shape. Figure 3-5 shows the images cr eated by extracting the intensity of m/z 756 [PC(16:0,16:0) + Na]+ divided by the intensity of m/z 588 for each strategy of internal standard application. The distinct H-pattern of gray matter is clearly obse rved in each image, and this signal appears to be consistent between tissue se ctions, which implies an even signal distribution of both the analyte and the intern al standard. However, the mo st well-defined tissue shape is seen in Figure 3-5C, application of internal standard under wet tissu e. The edges of the tissue as well as the gray matter pattern are less clearly de fined in the other two images, Figures 3-5A and 3-5B, which may suggest some migration of the an alyte. Areas of increased intensity, known as hot spots, are seen in all three images and are likely due to inhomogeneous crystallization of the MALDI matrix rather than an increased concentration of lipids in those areas. Quantitation Table 3-1 compares relative concentration of PCs of varying fatty acid composition from published12 results of extracted phospholipids in rat brain tissue with MS results acquired in this study from one section of rat brain and spinal cord. Phospholipids from the published results

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05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000ABC m 05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000ABC 05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 05001000150020002500300035004000 0 500 1000 1500 2000 2500 3000 3500 4000 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000ABC m700Figure 3-4. Mass spectral images of the [M+Na]+ ion of the internal standard, m/z 588, applied in a 3-mm square to serial sections of rat brain A) on top of dry ti ssue, B) on top of wet tissu e, and C) under wet tissue. Figure 3-4. Mass spectral images of the [M+Na]+ ion of the internal standard, m/z 588, applied in a 3-mm square to serial sections of rat brain A) on top of dry ti ssue, B) on top of wet tissu e, and C) under wet tissue.

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71Figure 3-5. Mass spectral images of the [M+Na]+ ion of PC(16:0,16:0), m/z 756, divided by the intensity of the internal standard, m/z 588, applied A) on top of dry tissue, B) on t op of wet tissue, and C) under wet tissue. Figure 3-5. Mass spectral images of the [M+Na]+ ion of PC(16:0,16:0), m/z 756, divided by the intensity of the internal standard, m/z 588, applied A) on top of dry tissue, B) on t op of wet tissue, and C) under wet tissue. 050010001500200025003000 0 500 1000 1500 2000 2500 3000 05001000150020002500 3000 0 500 1000 1500 2000 2500 3000 050010001500200025003000 0 500 1000 1500 2000 2500 3000 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 ABC m 050010001500200025003000 0 500 1000 1500 2000 2500 3000 05001000150020002500 3000 0 500 1000 1500 2000 2500 3000 050010001500200025003000 0 500 1000 1500 2000 2500 3000 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 ABC 050010001500200025003000 0 500 1000 1500 2000 2500 3000 05001000150020002500 3000 0 500 1000 1500 2000 2500 3000 050010001500200025003000 0 500 1000 1500 2000 2500 3000 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 ABC m

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Table 3-1. Literature and MS data comparison of relative concentration of phosphatidylcholines of varying fatty acid tail composition P hosphatidylcholineLiterature (Brain)MS (Brain) MS (Cord) ( SE) 14:0,16:0 3.1 1.1 0.1 ( 0.004) 16:0,16:0 19.214.3 4.6 ( 0.3) 16:0,18:1 36.233.3 37.5 ( 0.5) 16:0,20:4 4.4 5.4 0.7 ( 0.05) 18:0,18:1 14.115.6 20.5 ( 0.5) 18:0,20:4 3.8 8.7 2.5 ( 0.07) 18:1,18:1 3.4 5.9 18.3 ( 0.5) Reprinted with permission from Agranoff, B.W. et al Basic Neurochemistry: Molecular, Cellular and Medical Aspects. pp. 3349 (Lippincott-Raven, Philidelphia, 1998) 72

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73 were extracted from the homogenized forebrai n region and separated by TLC then hydrolyzed. The hydrolyzed fatty acids were analyzed by re verse phase HPLC for each lipid class and the fractions were collected, meth anoylized, and identified by GC. Mol% was calculated based on the addition of one intern al standard, 1,2-distearoyl-sn -glycerol. For this study, internal standard was applied on top of dry tissue and mol% was calculated from brain or spinal cord sections by averaging the spectra across the entire section and recording the intensity of each ion, as seen in Figure 3-6. One saggital brain section was analyzed once, and three spinal cord sections were analyzed three times. It was assumed that the MALDI response factor for each PC (including th e internal standard) is the same, not an unreasonable assumption within a single compound class, as here. Thus, the mol% can be calculated simply based on intensity% (just for PC s). The number of moles was calculated using Equation 3-1 moles = (IntAN)(IntIS)-1(WIS)(MWAN)-1(A) (3-1) where IntAN is the intensity of the analyte peak, IntIS is the intensity of the internal standard peak, WIS is the amount of internal standard applied (50 pg/mm2), MWAN is the molecular weight of the analyte, and A is the area of the tissue. Area can be measured from the image of the tissue. The sum of the total mol% stated in the literature for lipids chosen for comparison is 84.2%. Calculation of mol% was achieved by summing the number of moles for each ion and dividing individual mole counts by a sum to give a mol% total of 84.2%. As shown in Table 3-1, the mol% for the PC s from the MS data taken from the brain section shows a remarkable correlation to the lite rature values. A previous comparison based on relative abundance of lipid ions generated by MALDI has also shown similar results.18 A good correlation between data from the spinal cord and the brain is observed for a majority of the ions,

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74 Figure 3-6. Mass spectra of the phos pholipid region averaged over the entire se ction of A) rat brain a nd B) rat spinal cord 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 782.6 798.6 810.6 783.6 756.6 826.6 772.6 799.6 753.7 784.6 811.6 800.6 769.6 760.7 828.6 848.7 773.6 808.6 836.7 723.6 812.6 788.7 824.6 761.6 751.6 734.6 844.5 739.6 782.7 810.7 798.6 783.7 835.7 811.7 826.6 850.8 799.6 760.7 784.7 753.7 852.7 769.7 837.7 788.7 756.7 824.7 812.7 723.7 761.7 774.8 751.7 742.9A B 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 782.6 798.6 810.6 783.6 756.6 826.6 772.6 799.6 753.7 784.6 811.6 800.6 769.6 760.7 828.6 848.7 773.6 808.6 836.7 723.6 812.6 788.7 824.6 761.6 751.6 734.6 844.5 739.6 782.7 810.7 798.6 783.7 835.7 811.7 826.6 850.8 799.6 760.7 784.7 753.7 852.7 769.7 837.7 788.7 756.7 824.7 812.7 723.7 761.7 774.8 751.7 742.9 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 782.6 798.6 810.6 783.6 756.6 826.6 772.6 799.6 753.7 784.6 811.6 800.6 769.6 760.7 828.6 848.7 773.6 808.6 836.7 723.6 812.6 788.7 824.6 761.6 751.6 734.6 844.5 739.6 782.7 810.7 798.6 783.7 835.7 811.7 826.6 850.8 799.6 760.7 784.7 753.7 852.7 769.7 837.7 788.7 756.7 824.7 812.7 723.7 761.7 774.8 751.7 742.9A B

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75 brain section. For example, PC(18:0,18:1) and PC(18:0,20:4) are notably higher, while PC(14:0,16:0), PC(16:0,16:0), and PC(16:0,20:4) are lower. To the authors knowledge, no literature exists that reports the individual lipid content found in sp inal cord. More insight into these differences can be gained by comparing the mass spectra collected from the brain section and a spinal cord section, as shown in Figure 3-6. An attempt to quantitate other lipid cla sses found in nerve tissue presents a unique challenge. The preformed positive charge of the quaternary amine of PCs and SPMs enhances the detectability of these lipids ov er that of other lipid species.96 Identification of lipids through the use of MS/MS shows that the majority of i ons detected in positive ion mode is PCs, and rarely is a PE or PS detect ed as the dominant species.18,80 Refer also to Table 5-1 in Chapter 5. Therefore, a more reliable method for quantif ying low abundance ions wo uld be through MS/MS analyses. The MS/MS spectrum of m/z 856 can be seen in Figure 3-7. The spectrum is dominated by the ion at m/z 797 which is a neutral loss of 59 corresponding to the trimethylamine of a PC. The ions at m/z 813 an d 769 correspond to the neutral loss of 43 and 87 indicating the presence of a PE and a PS, respectively. The ions at m/z 673, 572, and 524 coincide to the neutral losses of the PC headgroup and fatty acid tails of the lipids present. Due to the presence of isobars, th e quantitation of low abundance species could be accomplished through MS/MS experiments; however, a separate MS/MS scan would have to be performed for the internal standard which would introduce greater uncertainty in the quantitation process.97 An alternative and promising technique for the quantitation of isobaric species is the analysis of tissue using high mass resolution. Mu ltiple internal standards could be applied to tissue, and one full scan would produce data for e ach internal standard and all the isobars of interest. An example of a highly resolved ma ss spectrum of the lipid region in spinal cord

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Figure 3-7. MS/MS spectrum of m/z 856 showi ng the neutral losses (NL) of 43, 59, and 87, which indicates the presence of isobar ic lipids. PE: [phosphatidylethanolamine (42:7) + K]+; PC: [phosphatidylcho line (18:0,22:6) + Na]+; PS: [phosphatidylserine (18:0,20:4) + 2Na]+ Figure 3-7. MS/MS spectrum of m/z 856 showi ng the neutral losses (NL) of 43, 59, and 87, which indicates the presence of isobar ic lipids. PE: [phosphatidylethanolamine (42:7) + K]+; PC: [phosphatidylcho line (18:0,22:6) + Na]+; PS: [phosphatidylserine (18:0,20:4) + 2Na]+ NL 59 PC NL 87 PS NL 43 PE NL 59 PC NL 87 PS NL 43 PE76 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 797.6 673.6 572.5 769.7 838.4 524.5 813.5 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 797.6 673.6 572.5 769.7 838.4 524.5 813.5 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z 797.6 673.6 572.5 769.7 838.4 524.5 813.5 90 80 70 60 50 40 100Relative Abundance30 20 10 0

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77 obtained on an orbitrap mass spectro meter is given in Figure 3-8. The inset is an expansion of the m/z range 856-857. A dramatic increase in resolution and decrease in background is noted when compared to Figure 3-6B. A change in relative abundance for se veral ions is also observed. These issues are discusse d in detail in Chapter 4, but noted here in these two spectra is the demonstration of ease with which an internal standard an d both high and low abundance ions could be monitored using high resolution scanning. Tissue Comparison A comparison of the relative abundance of seve n PCs from control and DCA-administered tissue is given in Table 3-2. While the addition of sodium acetate creates a mass spectrum that is dominated by ions that are sodium adducts, some m/z values are dominated by the potassium adduct. For those lipids that a ppear in the mass spectrum as different ionic species (for instance [M+Na]+ and [M+K]+), the relative intensities are summed. Data collected from spinal cord tissue of three control rats (C1.0, C1.2, C2.1) and three rats administer ed DCA (DCA5.1, DCA6.1, DCA7.1) are shown. Three 10-m serial sec tions were cut from the spinal cord of each animal, internal standard was applied on top of dry tissue, and the tissue was analyzed three times at a relative laser power of 30 with 10 la ser shots per raster step across the entire tissue section. An average section of spinal cord used in this study weighed less than 50 g. When compared to conventional techniques such as LC/MS,98 the amount of sample used here is at least an order of magnitude sm aller. The average precision am ong the control tissue is 4% SE and 3% SE among the DCA-administered tissue. An average decrease of about 4 fold was reported in Chapter 2 in the relative abundance of lipid ion intensities from DCA-administered tissue when compared to those intensities from control tissue; however, su ch a decrease is not observed here. Tissue from the previous study was reanalyzed and a decrease in ion intensity for

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78 Figure 3-8. Mass spectrum of the phospholipid range from spinal cord taken at 100,000 re solution. The inset is an expansion of the m/z region 856-857. 856.0 856.2 856.4 856.6 856.8 m/z 0 10 20 30 40 50 60 70 80 90 100 856.5863 856.4663 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 782.5706 810.6019 850.6771 798.5447 723.4970 832.6673 739.4712 826.5761 776.5966 751.5285 866.6515 767.5027 709.5178 856.0 856.2 856.4 856.6 856.8 m/z 0 10 20 30 40 50 60 70 80 90 100 856.5863 856.4663 856.0 856.2 856.4 856.6 856.8 m/z 0 10 20 30 40 50 60 70 80 90 100 856.5863 856.4663 856.0 856.2 856.4 856.6 856.8 m/z 0 10 20 30 40 50 60 70 80 90 100 856.5863 856.4663 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 782.5706 810.6019 850.6771 798.5447 723.4970 832.6673 739.4712 826.5761 776.5966 751.5285 866.6515 767.5027 709.5178 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 782.5706 810.6019 850.6771 798.5447 723.4970 832.6673 739.4712 826.5761 776.5966 751.5285 866.6515 767.5027 709.5178

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79Table 3-2. Comparison of relative concentration (SE) of phosphatidylcholines of varying fatty acid composition in spinal cord from three control (C) and three DCA-administerd animals PhosphatidylcholineC1.0C1.2C2.1DCA5.1DCA6.1DCA7.1 14:0,16:0 0.1 ( 0.003)0.1 ( 0.009)0.1 ( 0.003)0.1 ( 0.003)0.1 ( 0.002)0.1 ( 0.006) 16:0,16:0 3.8 ( 0.5)5.0 ( 0.2)3.3 ( 0.6)4.3 ( 0.1)5.4 ( 0.4)4.6 ( 0.4) 16:0,18:1 38.4 ( 0.5)37.9 ( 0.2)40.5 ( 0.3)38.3 ( 0.7)39.1 ( 1.0)40.6 ( 0.3) 16:0,20:4 0.7 ( 0.06)0.7 ( 0.02)0.5 ( 0.05)0.8 ( 0.03)0.9 ( 0.1)0.8 ( 0.03) 18:0,18:1 19.0 ( 0.5)18.9 ( 0.4)18.7 ( 0.3)18.3 ( 0.1)18.1 ( 0.3)17.6 ( 0.1) 18:0,20:4 19.5 ( 0.5)19.2 ( 0.5)18.7 ( 1.1)19.7 ( 0.8)17.9 ( 1.5)17.7 ( 0.7) 18:1,18:1 2.7 ( 0.1)2.5 ( 0.05)2.3 ( 0.03)2.8 ( 0.09)2.6 ( 0.1)2.7 ( 0.06)

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DCA-administered animals was still seen. The data presented in this chapter were obtained from a different set of test subject s than the data obtained for the study in Chapter 2. Tissue preparation and handling is bei ng investigated, but no evidence has been found that may explain the discrepancies between the data. Nonetheless, highly reproducible results were obtained in the present study. Conclusions The ability to quantitate endogenous lipids in nerve tissue was demonstrated in the research presented here. First, signal intensity was monitored in successive analyses of a single tissue section to provide insight into the variation of signal res ponse with an increasing number of laser shots. Such an experi ment is necessary to minimize erro rs due to signal variability and for direct comparisons to other MS and quantitative techniques. A non-endogenous phospholipid, PC( 10:0,10:0) was explored fo r use as an internal standard. It was chosen because it does not inte rfere with ions typically found in the lipid mass range (m/z 700 900) as well as having similar ionization and extraction efficiency to the endogenous lipids of interest. Thr ee different application methods of the internal standard were evaluated, on top of dry tissue, on top of wet tissue, and under wet tissue. For quantitative purposes, it was determined that application of the internal sta ndard on top of dry tissue provided the most even coating and signal distribution. Ho wever, generation of tissue images shows that the greatest spatial resolution and most accurate anatomical structure are observed by applying internal standard under wet tissue. Finally, endogenous lipids were quantified in rat brain and sp inal cord tissue sections. Results showed that quantitation performed by MALDI were similar when compared to published results. Sample preparation for MALDI is relatively less tedious than for traditional quantitative methods such as LC/MS and only requir es micrograms of sample. The quantitative 80

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results displayed here illustrate the potential of this technique to provide a rapid and reliable means of quantitation 81

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CHAPTER 4 IMAGING OF LIPIDS IN SPINAL CORD USING INTERMEDIATE PRESSURE MALDILIT/ORBITRAP MS Introduction The direct interrogation of sec tioned tissue has piqued the intere st of scientists worldwide due to the wide range of molecular species that can be analyzed and the ability to spatially monitor each one. Conventional analyses have b een focused on the distri bution of proteins and peptides,99-102 drugs and metabolites,36,103,104 and lipids,105-107 which spans a rather broad mass range. To encompass this range, tim e-of-flight (TOF) mass spectrometers108-110 have typically been employed as the mass analyzer of choice. Not only does TOF/MS provide a wide mass range, it offers rapid scan speeds, mass re solution around 10,000, and mass accuracy below 20 ppm.111 Improved mass resolution and accuracy have become a center of interest in mass spectrometric imaging as a result of the detection of isobaric species in tiss ue, particularly in the low mass region. Along with endogenous compounds, matrix clusters present as isobars and add to the complexity of data analysis. High-performance instruments, such as Fourier transform ion cyclotron resonance (FTICR), offer high-resolu tion scanning (R > 100,000) capable of resolving many ions in a very narrow m/z range (< 1 amu) in a single experiment.79,85 Along with low ppm mass accuracy, FTICR demonstrates a methodol ogy highly desirable for the analysis of tissue. However, the technique is hindered by in herently slow scan speeds and often laborious identification of unknowns based solely on accurate mass. Both TOF/MS and FTICR/MS extend advantag es for tissue imaging, but typically offer only one stage of mass analysis. To circumvent this drawback, the addition of an external collision cell79,85 is often utilized on an FTICR to yield MS/MS data that aid in identification. However, this contributes to extended analyses times. MS/MS spectra are also obtained by 82

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isolating the precursor ion in the ICR cell and exciting it with a pulsed laser,112,113 but fragment energies are often low. TOF/MS renders a mo re compatible design to the implementation of MS/MS analyses. Post source decay114,115 is often used due to the ease of execution, but lacks the ability to control fragmentation. TOF/TOF116,117 and qTOF118,119 instruments have both been proven to successfully produce MS/MS data, but again, yield relatively low resolution mass spectra and a high degr ee of fragmentation. In this study, we describe the use of a hybr id linear ion trap/orbit rap mass spectrometer with intermediate pressure MALDI for the imaging of lipids in spinal cord. The linear ion trap provides MSn capabilities, and the orbitrap can pr ovide mass resolution of 100,000. This new design affords the ability to identify ions based on MSn fragmentation and accurate mass and to image the thousands of peaks detected in a sing le, high-resolution scan as well as high-resolution daughter scans. Experimental Animal Treatment and Tissue Preparation All animal studies were approved by the loca l IACUC at UCSD and were performed in an AAALAC-approved vivarium. Adult (250-300g body weight) female Sprague-Dawley rats (Harlan, San Diego, CA) were housed 3 per cage under a 12 hr light:dark cycle with free access to food (Harlan Teklad 7001) and water. Exci sed tissue was immediately flash frozen in liquid nitrogen and stored at -80C until shipped on dry ice in plastic tubes to the University of Florida, where it was again stored at -80 C until processed. Frozen tissue was sectioned to a thickness of 10 m at a temperature of -25C using a Leica CM1850 cryotome (Houston, TX). The tissue was attached to the cryotome sample stage usi ng water to avoid the ma ss spectral interferences caused by traditional techniques th at use polymers to affix the ti ssue to the sample stage. 83

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84 Matrix Application Sectioned tissue was thaw-mounted on glass microscope slides and then dried in a vacuum desiccator for 30 minutes. The matrix, 40mg/ml 2,5-dihydroxybenzoic acid (DHB) in 70:30 methanol:water with 10-mM sodium acetate, wa s applied using an Epson R260 inkjet printer.20 The microscope slides were placed in a modified CD tray holder and passed through the printer 15 times for matrix application. Parameters in th e Epson CD printing software were adjusted to give an even coat of matrix across the entire tissue section. Evenness was monitored visually using a dissecting microscope with transm itted illumination through a blue filter. Instrumentation Studies were carried out on a hybrid linear ion trap/orbitrap mass spectrometer fitted with a MALDI ion source that operates at intermediate pressure (MALDI LTQ Orbitrap XL, Thermo Fisher Scientific, Bremen, Germany). Briefly, a N2 laser with a wavelength of 337 nm is focused directly to the source, which opera tes at intermediate pressure of about ~100 mTorr. As seen in Figure 4-1, the generated MALDI-ions are then tr ansferred through a seri es of differentially pumped quadrupole and octapole ion guide s to a linear ion trap where MSn analysis can be performed or the ions are passed on to the C-trap. Ions that are transmitted to the C-trap are collisionally cooled there before injection into th e orbitrap for detection or into the HCD cell for fragmentation. The use of nitrogen at higher pressures in the HCD cell creates higher energy collisions than those typically found in the linear ion trap whic h leads to a greater number of fragment ions. The octapole geometry also allo ws for a lower mass cut off, making detection of low-mass ions possible. Fragment ions produced in the HCD cell are transferred back to the Ctrap and injected into the orbitrap for detection.

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Figure 4-1. Hybrid linear ion trap /orbitrap with intermediate pressure MALDI source adapted from MALDI LTQ Orbitrap product specifications from Thermo Fisher Scientific. Red lines i ndicate stable ion trajectories. Ion guides labeled q and o correspond to quadrupoles and octaples, respectively. Figure 4-1. Hybrid linear ion trap /orbitrap with intermediate pressure MALDI source adapted from MALDI LTQ Orbitrap product specifications from Thermo Fisher Scientific. Red lines i ndicate stable ion trajectories. Ion guides labeled q and o correspond to quadrupoles and octaples, respectively. 85 OrbitrapMass Analyzer MLDI Source Linear Ion Trap C-TrapHCD Collision Cellq qo o oLaser Beam A OrbitrapMass Analyzer MLDI Source Linear Ion Trap C-TrapHCD Collision Cellq qo o oA OrbitrapMass Analyzer MLDI Source Linear Ion Trap C-TrapHCD Collision Cellq qo o oLaser Beam A

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86 Tissue Analysis Microscope slides affixed with coated tissu e were adhered to a modified MALDI sample plate using copper tape. The laser raster size was set to 100 m. All experiments were performed with automatic gain control turned on, and the signal was optimized by manually adjusting the laser power fire d on each tissue specimen. Full-scan MS experiments were typically run at a laser power of 15 20 J. MS2 experiments required the same laser power, with AGC adjusting for the number of laser shots per step. Qualitative data were obtained using standard Xcalibur software and ImageQuest im aging software. Instrument calibration was performed twice a day using a peptide mi xture for both normal and high mass operation. Results and Discussion Orbitrap vs. Linear Ion Trap Imaging in our lab has typically employed a linear ion trap (LIT ) for mass analysis. It has proven successful for a wide ra nge of compounds and has been invaluable for identification purposes due to its MSn capabilities. However, LITs provide only unit resolution and mass accuracy of 100 ppm; often highe r resolution and mass accuracy would be helpful in detecting, identifying, and imaging compounds in complex tissue matrices. Figure 4-2 shows mass spectra of the phospholipid range taken from the same section of rat spinal cord generated from an orbitrap (4-2A) and an LIT (4-2B). The most notable differences between the spectra are the higher re solving power (R = 100,000 for the orbitrap vs. R = 4,000 for ion traps) and reduction of chemi cal noise achieved by the orbitrap. While unit resolution is attained on the LIT, low-abundance ions are ma sked due to the presence of MALDI matrix ions in the chemical background. Distin guishing ions originating from tissue from those arising from the MALDI matrix is virtually impossible for those th at have a signal intensity of less than 10% of the base peak. Examinati on of the relative abundance of the major ions

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87Figure 4-2. Mass spectra of the phosphlipid m/z region of a section of rat spinal cord. Each spectrum represents the same area of spinal cord tissue analyzed by A) an orbitrap and B) a linear ion trap. Relative Abundance 0 10 20 30 40 50 60 70 80 90 100 782.5706 798.5447 810.6018 850.6770 723.4970 832.6672 739.4712 776.5965 826.5761 751.5285 835.67028 6 6 6 5 1 5822.6465 753.5916 767.5027 760.5889 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.6 810.7 835.7 798.6 848.8 826.5 753.7 723.6 776.6 768.7 760.6 866.5 739.7A BRelative Abundance 0 10 20 30 40 50 60 70 80 90 100 782.5706 798.5447 810.6018 850.6770 723.4970 832.6672 739.4712 776.5965 826.5761 751.5285 835.67028 6 6 6 5 1 5822.6465 753.5916 767.5027 760.5889 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.6 810.7 835.7 798.6 848.8 826.5 753.7 723.6 776.6 768.7 760.6 866.5 739.7Relative Abundance 0 10 20 30 40 50 60 70 80 90 100 782.5706 798.5447 810.6018 850.6770 723.4970 832.6672 739.4712 776.5965 826.5761 751.5285 835.67028 6 6 6 5 1 5822.6465 753.5916 767.5027 760.5889 0 10 20 30 40 50 60 70 80 90 100 782.5706 798.5447 810.6018 850.6770 723.4970 832.6672 739.4712 776.5965 826.5761 751.5285 835.67028 6 6 6 5 1 5822.6465 753.5916 767.5027 760.5889 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.6 810.7 835.7 798.6 848.8 826.5 753.7 723.6 776.6 768.7 760.6 866.5 739.7 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.6 810.7 835.7 798.6 848.8 826.5 753.7 723.6 776.6 768.7 760.6 866.5 739.7A B

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between the two spectra shows great similarity bu t also exhibits evident differences, notably the increased abundance in the orbitrap spectru m of nominal m/z values 739, 798, and 866. The major ions at these m/z values have b een identified as [PC(16:0,18:1) + K N(CH3)3]+, [PC(16:0,18:1) + K]+, and [Cer(d18:1,24:0h) + K]+, respectively. Each ion is detected as a potassiated cation. Studies have sh own that the alkali metal adducts of lipid ions are more stable in the gas phase than the corresponding protonated species.18 The lengthened storage time of ions in the orbitrap compared to the storage time of a LIT (an order of magnitude difference) may cause loss of the less stable protonated i ons while an increased relative abundance is observed for cation species due to greater gas-phase stability. An advantage of reduced chemical noise is illustrated in Fi gure 4-3. The mass spectrometric images were generated by mapping the total ion current for serial sections of rat spinal cord for data collected on an orbitrap (4 -3A) and a LIT (4-3B). A clear tissue shape is observed for both images; the orbitrap data, however shows little or no signal detected off tissue and a distinct gray matter pattern seen in the center of the image. The greatest signal detected in the image created by the LIT is in the area off tis sue, where only DHB matrix is present. The dominant ion in a DHB spectrum is the M+ ion at m/z 154. However, DHB readily looses hydrogen and forms adducts with itsel f and water to form a host of cluster ions that appear at nearly every m/z value in the MALDI spectrum. Once ionization occurs, these matrix cluster ions, along with the analytes of interest, traverse a distance of ~17 cm (two quadrupole ion guides and a set of octapole rods) before reachin g the LIT, where they are trapped using helium as a bath gas. This distance is ~2 times shor ter than the distance an ion must travel (~40 cm) through the LIT and another octapole and the C-trap to reach the orb itrap. In addition, ions are trapped and collisionally cooled with nitrogen once they are injected into the C-trap. The 88

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0 100% 1 mm 1 mmAB 0 100% 0 100% 1 mm 1 mm 1 mm 1 mmABFigure 4-3. Mass spectrometric images of the total i on current of serial rat spinal cord sections analyzed by A) an orbitrap and B) a linear ion trap 89

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90 combination of a longer distance tr avelled, a longer traverse time, and more energetic cooling in the C-trap may lead to greater dissociation of the matrix cluste r ions. Furthermore, the long trapping time (~1 s) required for detection in the orbitrap may re sult in dissociation of loosely bound matrix cluster ions causing further reduction in MALDI matrix background. Another example of the benefit of reduced b ackground can be seen in Figure 4-4; these mass spectra were collected from the serial sectio ns imaged in Figure 4-3 obtained on an orbitrap (4-4A, 4-4B) and a LIT (4-4C, 4-4D) taken from white (4-4A, 4-4C) and gray (4-4B, 4-4D) matter. The mass spectral differences between white and gray matter become much more evident with the background reduction observe d in the orbitrap spectra, which could be beneficial to studies tracking cha nges in specific regions of tissue. Two such ions that exhibit dominance in the differing regions of sp inal cord are m/z 756 [PC(16:0,16:0) + Na]+ in gray matter and m/z 776 [PE(18:0,20:0) + H]+ in white matter, which are in agreement with literature values that demonstrate a greater abundance of PCs than PEs in gray matter and a greater abundance of PEs than PCs in white matter.12 The images of these ions are shown in Figure 4-5. Figures 4-5A and 4-5C are the images of m/z 756 created from data taken on an orbitrap and LIT, respectively. Increased si gnal is not only seen in the white matter section of Figure 4-5C but off tissue as well. A similar effect is perceived in the images of m/z 776. Greater signal is seen in the gray matter section and off tissue in the LIT image (4-5D) than in the orbitrap image (4-5B). These images provide examples of th e increased specificity possible with greatly reduced MALDI matrix background. Imaging using High Resolution MS Scanning Mass spectrometric images created from data obtained on mass analyzers that provide unit resolution have provided valuable insight into compound distribution within tissue;34,73,120 just as images generated by MS/MS can help differentiate isobaric compounds,34 so can high resolution

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91 Figure 4-4. Mass spectra of phospholip ids found in white matter (A,C ) and gray matter (B,D) analyzed on (A,B) an orbirtrap and (C,D) a linear ion trap. Figure 4-4. Mass spectra of phospholip ids found in white matter (A,C ) and gray matter (B,D) analyzed on (A,B) an orbirtrap and (C,D) a linear ion trap. 0 10 20 30 40 50 60 70 80 90 100 782.5703 850.6768 810.6016 723.4968 798.5445 832.6669 776.5963 751.5282 826.5758 866.6514 739.4710 767.5025 0 10 20 30 40 50 60 70 80 90 100 782.5707 798.5448 739.4712 723.4972 810.6022 826.5762 850.6780 772.5292 756.5554 866.6518 844.5292AB C 720 740 760 780 800 820 840 860 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.7 798.6 810.7 756.7 772.7 826.6 848.7 832.7 856.68 74 5742.87 2 3 7D 0 10 20 30 40 50 60 70 80 90 100 782.6 810.6 798.5 835.7 826.5 851.6 769.6 753.6 723.5 864.7 720 740 760 780 800 820 840 860 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.5703 850.6768 810.6016 723.4968 798.5445 832.6669 776.5963 751.5282 826.5758 866.6514 739.4710 767.5025 0 10 20 30 40 50 60 70 80 90 100 782.5703 850.6768 810.6016 723.4968 798.5445 832.6669 776.5963 751.5282 826.5758 866.6514 739.4710 767.5025 0 10 20 30 40 50 60 70 80 90 100 782.5707 798.5448 739.4712 723.4972 810.6022 826.5762 850.6780 772.5292 756.5554 866.6518 844.5292 0 10 20 30 40 50 60 70 80 90 100 782.5707 798.5448 739.4712 723.4972 810.6022 826.5762 850.6780 772.5292 756.5554 866.6518 844.5292AB C 720 740 760 780 800 820 840 860 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.7 798.6 810.7 756.7 772.7 826.6 848.7 832.7 856.68 74 5742.87 2 3 7 720 740 760 780 800 820 840 860 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.7 798.6 810.7 756.7 772.7 826.6 848.7 832.7 856.68 74 5742.87 2 3 7D 0 10 20 30 40 50 60 70 80 90 100 782.6 810.6 798.5 835.7 826.5 851.6 769.6 753.6 723.5 864.7 720 740 760 780 800 820 840 860 880 m/z 0 10 20 30 40 50 60 70 80 90 100 782.6 810.6 798.5 835.7 826.5 851.6 769.6 753.6 723.5 864.7 0 10 20 30 40 50 60 70 80 90 100 782.6 810.6 798.5 835.7 826.5 851.6 769.6 753.6 723.5 864.7 720 740 760 780 800 820 840 860 880 m/z

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0 100% 0 100% 1 mm 1 mmAB CD 1 mm 1 mm 1 mm 1 mmAB CDFigure 4-5. Mass spectrometric images of m/z 756 (A,C) and m/z 776 (B,D) generated from serial rat spinal cord sections analyzed on (A,B) an orbitrap and (C,D) a linear ion trap. The ion at m/z 756 is dominant in the gray matter of the spinal cord where the ion at m/z 776 is dominant in the white matter. 92

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93 MS images, as shown here. Figure 4-6 shows data obtained on an orbitrap. The image seen in Figure 4-6B was created by mapping a range of m/z values centered at m/z 844.5 amu, plus or minus 0.5 amu. This is the procedure used to produce images from data obtained on a LIT with unit mass resolution. By examining the highly resolved mass spectrum in Figure 4-6A, one can clearly see that more than one ion is detected at nominal mass 844; mapping a range of values produces an image dominated by the base peak of that m/z range, m/z 844.5292. Figures 4-6C 4-6E are images crated by mapping the individual ions at m/z 844.4690 0.02, 844.5292 0.02, and 844.9463 0.002, respectively. The two ions at m/z 844.4690 [PS(16:0,20:4) + Na + K]+ and 844.5292 [PC(16:0,22:6) + K]+ appear in the gray matter of the spinal cord; however, the ion at m/z 844.4690 has a greater distribution throughout all the gray matter, where the ion at m/z 844.5292 appears to be less abundant in the dorsal horns. The image of m/z 844.9463 (unidentified) is opposite of the other two ions and appears only in white matter. This ion is completely masked when imaging a range of values (4-6B), which illustrates the importance of high resolution analysis of comp lex samples such as tissue. Role of HRMS and MS/HRMS in Identifying Lipids The identification of ions based solely on accurate mass is a tedious process due to the immense number of elemental compositions comprisi ng the ions detected in nerve tissue. First, mass accuracy must be considered to give an accurate representation of the elemental composition. For example, two dominant peaks are detected at nominal mass 832 which are 832.5865 and 832.6671 and have been identified as [PC(18:0,20:4) + Na]+ and [Cer(d18:1,24:1) + Na]+, respectively. Accurate mass calculations of the two ions produce a mass of 832.5832 (2.9 ppm error) for the PC and 832.6642 (3.3 ppm erro r) for the Cer. The difference in masses for the observed values is 0.0806, and the differen ce in the calculated values is 0.0810 (0.4 ppm error).

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844.5 0.5 844.4690 0.02 844.5292 0.02 844.9463 0.0294 Figure 4-6. A) Mass spectrum of m/z regi on 844-845 showing at least 5 peaks are detected. The mass spectrometric images correspond to B) the 1 amu mass range and the peaks at C) 844.4690, D) 844.5292, and E) 844.9463. 844.0 844.1 844.2 844.3 844.4 844.5 844.6 844.7 844.8 844.9 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 844.529 2 844.9463 844.4690 844.0 844.1 844.2 844.3 844.4 844.5 844.6 844.7 844.8 844.9 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 844.529 2 844.9463 844.4690 1 mm 0 100%A BCDE844.5 0.5 844.4690 0.02 844.5292 0.02 844.9463 0.02 844.0 844.1 844.2 844.3 844.4 844.5 844.6 844.7 844.8 844.9 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 844.529 2 844.9463 844.4690 844.0 844.1 844.2 844.3 844.4 844.5 844.6 844.7 844.8 844.9 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 844.529 2 844.9463 844.4690 1 mm 0 100%844.5 0.5 844.4690 0.02 844.5292 0.02 844.9463 0.02 844.0 844.1 844.2 844.3 844.4 844.5 844.6 844.7 844.8 844.9 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 844.529 2 844.9463 844.4690 844.0 844.1 844.2 844.3 844.4 844.5 844.6 844.7 844.8 844.9 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 844.529 2 844.9463 844.4690 1 mm 1 mm 0 100% 0 100%A BCDE

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95 If determining the elemental composition of these two ions is attempted with no limitations, a list is generated th at contains hundreds of possibl e combinations. By applying a constraint to include only those combinations within the mass error, three possibilities are given for the Cer and thirteen are given for the PC. Of these likely combinations, only two from each list are reasonable for lipid ions. However, th ere is no distinction between isomeric compounds. A Cer would not have any isomers because of the lack of a phosphorus atom, but a PE ion with a fatty acid composition of (41:4) has the same molecular formul a as a PC with a fatty acid composition of (38:4). While odd chained fatty acids are not comm on, the possibility of a lipid containing such a fatty acid can not be ignor ed nor confirmed without the use of MS/MS. MS/MS has proved to be invaluable for the identification and differentiation of isobaric/isomeric species. Daughter ions in the MS/MS spectrum have allowed identification of lipid sub-classes based on characteristic fragme ntation (i.e. NLs of 43, 59, and 87 for PEs, PCs, and PSs, respectively) as well as fatty acid ta il assignment based on relative intensity of the fragment. Images generated from these characteristic fragments show distinct differences in distribution within the tissue.34 The roles of high mass accuracy and MS/M S were used here to provide highly dependable identification of lipid ions. An expa nsion of the m/z range 848 849 on the orbitrap spectrum in Figure 4-2A can be seen in Figure 4-7A. At least four ions are detected within this mass range; the images corresponding to m/z 848.5603, 848.6417, and 848.6617 are shown in 47B 4-7D. The ion at m/z 848.6975 is too low in abundance to produce a useful image. The distribution of ions in Figures 4-7C and 4-7D are very similar within the white matter, whereas the ion in Figure 4-7B is dist ributed only in the gray matter.

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789.4846 484.326096 Figure 4-7. A) Mass spectrum of m/z region 848-849 acquired on an orbitrap. Mass spect rometric images B-D correspond to major ions detected in A. Fragmentation of nominal m/z 848 in the linear ion trap produces the MS/MS spectrum seen in E. Images observed in F and G correspond to the two major isobars found at 848. Matc hing images produced by the full scan and MS/MS spectra and exact mass calculations identif y the ions in the full scan mass spectrum as: 848.5603 [PC(18:0,20:4) + K]+; 848.6417 [Cer(d18:1,24:1) + K]+; 848.6617 [Cer(d18:1,24:1h) + Na]+; 848.6975 [Cer(d18:1,25:0) + Na]+. NL: neutral loss; PC: phos phatidylcholine; PS: phosphatidylserine; Cer: cerebroside 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208NL 18 NL 59 PC NL 162 Cer NL 180 Cer NL 87 PS Spingosinealdehydebase Cer Spingosinebase Cer 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208NL 18 NL 59 PC NL 162 Cer NL 180 Cer NL 87 PS Spingosinealdehydebase Cer Spingosinebase Cer 848.5603 848.6417 848.6617 848. 2 848.3 848.4 848.5 848.6 848.7 848.8 848.9 849.0 m/z 0 20 40 60 80 100Relative Abundance 848.6617 848.6417 848.5603 848.6975 848. 2 848.3 848.4 848.5 848.6 848.7 848.8 848.9 849.0 m/z 0 20 40 60 80 100Relative Abundance 848.6617 848.6417 848.5603 848.69751 mm 0 100%A EB C D F G789.4846 484.3260 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208NL 18 NL 59 PC NL 162 Cer NL 180 Cer NL 87 PS Spingosinealdehydebase Cer Spingosinebase Cer 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208NL 18 NL 59 PC NL 162 Cer NL 180 Cer NL 87 PS Spingosinealdehydebase Cer Spingosinebase Cer 848.5603 848.6417 848.6617 848. 2 848.3 848.4 848.5 848.6 848.7 848.8 848.9 849.0 m/z 0 20 40 60 80 100Relative Abundance 848.6617 848.6417 848.5603 848.6975 848. 2 848.3 848.4 848.5 848.6 848.7 848.8 848.9 849.0 m/z 0 20 40 60 80 100Relative Abundance 848.6617 848.6417 848.5603 848.69751 mm 0 100%789.4846 484.3260 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208NL 18 NL 59 PC NL 162 Cer NL 180 Cer NL 87 PS Spingosinealdehydebase Cer Spingosinebase Cer 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208 40 0 450 500 550 600 650 700 750 800 850 m/z 0 20 40 60 80 100Relative Abundance 484.3260 686.6062 848.6602 668.5962 789.4846 830.6493 512.3208NL 18 NL 59 PC NL 162 Cer NL 180 Cer NL 87 PS Spingosinealdehydebase Cer Spingosinebase Cer 848.5603 848.6417 848.6617 848. 2 848.3 848.4 848.5 848.6 848.7 848.8 848.9 849.0 m/z 0 20 40 60 80 100Relative Abundance 848.6617 848.6417 848.5603 848.6975 848. 2 848.3 848.4 848.5 848.6 848.7 848.8 848.9 849.0 m/z 0 20 40 60 80 100Relative Abundance 848.6617 848.6417 848.5603 848.69751 mm 0 100% 0 100%A EB C D F G

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Isolation of the ions at m/z 848.5 .5 in the linear ion trap and subsequent fragmentation in the LIT produces the orbitrap MS/MS spectrum given in Figure 4-7E. The daughter ion at m/z 830.6493 arises from a neutral loss (NL) of 18 (water) and is a common fragment among biomolecules. The daughter ion at m/z 789.4846 is a NL of 59 and arises from the loss of the choline head group, N(CH3)3, from a PC. A low abundance daughter ion at m/z 761.4536 arises from a NL of 87 and is consistent with the loss of the serine head group (C3H5NO2) of a PS ion. The daughter ions at m/z 686.6062, 668.5962, 512.5208, and 484.3260 all correspond to losses associated with a Cer ion. The NL of 162 (m/z 686.6062) is a loss of C6H10O5 from the head group, the NL of 180 (m/z 668.5962) is a loss of the galactose head group, m/z 512.5208 is the loss of the fatty acid tail after the carbonyl, and m/z 484.3260 is the loss of the entire fatty acid tail. The image created using daughter ion of m/z 848 at 789.4846 (Fi gure 4-7F) matches the pattern of the image of the fu ll scan m/z 848.5603 (Figure 4-7B), which indicates that the latter m/z value corresponds to a PC. Performing an exact mass calculation (848.5572) allows this ion to be identified as [PC(18:0,20:4) + K]+. The images produced from the four Cer daughter ions all exhibit the same pattern, so only the image created by m/z 484.3260 (Figure 4-7G) was chosen for comparison. The pattern matches that of the images for the m/z values 848.6417 and 848.6617 (Figures 4-7C and 4-7D) in the full-scan mass spectrum. This suggests that both these isobaric ions could be Cers. Th e daughter ion arising from the NL of 87 is too low in abundance to give a useful image; however, the calculated exact mass for a PS (mos t likely [PS(18:1,18:1) + Na + K]+) would be detected at 848.4812, which elimin ates the possibility of the ion at m/z 848.6417 being a PS. Several possibilities exist for the identific ation of Cers due to the possible hydroxylation of the C2 carbon in the fatty acid. The NL of ~364 between the parent ions at mass 848.6412 or 97

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848.6617 and 484.3260 suggests a fatty acid with eith er 24 or 25 carbons. By calculating exact masses, three Cers can be identified as ma tches to nominal mass 848: [Cer(d18:1,24:1) + K]+ 848.6382; [Cer(d18:1,24:1h) + Na]+ 848.6592; [Cer(d18:1,25:0) + Na]+ 848.6955. The exact masses for these three ions have mass discrepancies of 0.0035, 0.0025, and 0.0020, respectively, from the masses detected in the full-scan mass spectrum (848.6417, 848.6617, and 848.6975). The mass discrepancy for [PC(18:0,20:4) + K]+ detected at m/z 848.5603 is 0.0031. The mass error for all the ions is within th e range of that discussed previously in this section. Therefore, a high degree of confidence in correct identification is maintained. Conclusions The utility of imaging mass spectrometry us ing a hybrid LIT/orbitrap mass spectrometer for analysis of lipids in spinal cord is clearl y demonstrated, and results showed that each mass analyzer on its own provides a unique way of analyzing the wealth of information obtained. While each mass analyzer can stand alone for imaging MS, with MS/MS provided by the LIT and HRMS by the orbitrap, combin ing the two, provides a superior method for the analysis of complex tissue. Full-scan mass spectra and ma ss spectral images were comp ared between data acquired on an orbitrap and a LIT. The increased mass spectral resolution on the orbitrap provided mass spectra with substantially le ss chemical background. The reduction in background made observation of variations in composition in different tissue regions more easily distinguishable. Background reduction was also clearly evident in mass spectrometric images, which enabled a more accurate visualization of the anatomical distribution of lipids. A comparison of MS images was also ma de between data produced from each mass analyzer. The images generated frm the LI T encompassed a 1 amu wide window corresponding to unit mass resolution. This method gives an acc urate representation of the ion distribution of 98

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the most intense ion within the 1 amu window. Analysis of the same 1 amu window by the orbitrap with mass resolution of 100,000 showed several ions each baseline-resolved. Mass accuracy was typically ~3 ppm. Images of each ion generated with a 0.02 amu window showed diverse distributions within the tissue sample. Lastly, identification of ions was achieve d by employing both mass analyzers. Orbitrap, full-scan HRMS images were matched to LI T/orbitrap MS/HRMS images; Assignment of daughter ion accurate mass and neutra l losses allowed for rapid iden tification. The ability of the LIT/orbitrap to provide both MS/MS (and MSn) and accurate mass HRMS data (on both MS and MS/MS spectra) made it an ideal system for imaging tissue. 99

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CHAPTER 5 IDENTIFICATION OF LIPIDS IS NERVE TISSUE: A COMPARISON OF IONIZATION METHODS USING A HYBRID LTQ/ORBITRAP Introduction Lipids are major components of boimembranes and serve as a host to numerous biological functions such as storage, cel lular structure, and signaling.121 For this reason, they have become of great interest to the scientific community. Lipi ds have a basic structure that typically contains a backbone with a polar head gr oup and a non-polar, fatty acid moiety. Two major categories of lipids are glycerophospholipids and sphingolipids. Phospholipids c onsist of a phosphate as part of the head group and two fatty acid tails that can vary in carbon length and degrees of unsaturation. Further classification is base d on the varying head groups attached to the phosphate. Sphingolipids contain a fixed 18-carbon sphingosine or sphingonine base and one varying fatty acid tail as well as varying polar head groups. The investigation of lipids by mass spectrome try has rapidly gained popularity over the past decade, with only 83 articles be ing published in 1998 to almost 250 in 2007.122 The coupling of soft ionization tec hniques, most specifically electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (M ALDI), to the highly sensitive detection capabilities of mass spectrome try has created an ideal methodology for the study of these biologically significant compounds. Both ESI98,123,124 and MALDI125-127 have been successfully employed for the analysis of lipid mi xtures and tissue extracts, and MALDI27,108,128-130 has demonstrated the ability to interrogate lipid spec ies directly from tissue. Typical experiments involving ESI often utilize liquid chromatograp hy for cleanup and separation and ion trap131,132 or triple quadrupole133,134 mass analyzers to perform tande m mass spectrometry, which not only provides further reduction of interf erences but also allows lipid class identification and fatty acid tail assignment. MALDI-TOF135,136 and MALDI-TOF/TOF90,92 have routinely been used 100

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because of rapid analysis times and accurate mass capabilities. MALDI is also known to be more tolerant of the high salt c ontent of tissue. More recentl y, lipid analysis by MALDI has been carried out on a linear ion trap34,73 permitting both fast analysis times and identification based on MSn measurements. In the present study, lipid extracts were analyzed by direct infusion nanospray/MSn and MALDI/MSn. The direct analysis of tissue by MALDI/MSn was also performed. The mass spectrometer employed was a hybrid LTQ/orbitrap that provided MSn capabilities as well as mass spectral resolution of 100,000. The utilization of three different, yet complementary, ionization techniques in positive ion mode identif ied five classes of lipids which include the glycerophospholipids phosphatidyalcholines (P C), phosphatidylethanolamines (PE), and phosphatidylserines (PS), as well as the sphingo lipids sphingomyelins (SPM) and cerebrosides (Cer). A majority of the ions formed from these lipid species are present as isobars, however; tandem MS and high resolution mass spectra made identification of these ions possible. Experimental Animal Treatment All animal studies were approved by the loca l IACUC at UCSD and were performed in an AAALAC-approved vivarium. Adult (250-300g body weight) female Sprague-Dawley rats (Harlan, San Diego CA) were housed 3 per cage under a 12 hr light:dark cycle with free access to food (Harlan Teklad 7001) and water. Exci sed tissue was immediately flash frozen in liquid nitrogen and stored at -80C until shipped on dry ice in plastic tubes to the University of Florida, where it was again stored at -80 C until processed. 101

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Tissue Preparation Intact tissue Frozen tissue was sectioned to a thickness of 10 m at a temperature of -25C using a Leica CM1850 cryotome (Houston, TX). The tissue was attached to the cryotome sample stage using water to avoid the mass spectral interfer ences caused by traditional techniques that use polymers to affix the tissue to the sample stag e. Sectioned tissue was thaw-mounted on glass microscope slides and then dried in a vacuum desiccator for 30 minutes. The matrix, 40-mg/ml 2,5-dihydroxybenzoic acid (DHB) with 10-mM s odium acetate, was applied using an Epson R260 inkjet printer.20 The microscope slides were placed in a modified CD tray holder and passed through the printer 15 times for matrix application. Parameters within the Epson CD printing software were adjusted to give an even coat of matrix across th e entire tissue section. Evenness was monitored visually using a dissecting microscope with transmitted illumination through a blue filter. Tissue extracts Lipids were extracted from a 1-cm piece of sciatic nerve (~1 mm diameter) and 120m section of spinal cord (~4 mm diameter). The tissue was homoge nized and an internal standard, phophatidylcholine(12:0,12:0), was added to the homogenate before extraction by the Folch method.137 The final residue was reconstituted in 500l of isopropanol. For direct infusion experiments, 250l of the extract diluted wi th 1.25 ml of methanol/isop ropanol/water (70/20/10) with 0.1% trifluoroacetic acid. A stainless st eel MALDI well plate was prepared by mixing a 50/50 solution of the extract with DHB matrix and spotting 1l aliquots into individual wells. Instrumentation Studies were carried out on a hybrid linear ion trap/orbitrap mass spectrometer fitted with either a nanospray source, or a MALDI ion sour ce that operates at intermediate pressure 102

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(Thermo Fisher Scientific, Bremen, Germany). Ions generated by nanospray or MALDI are transferred through a series of differentially pumpe d quadrupole and octapole ion guides into an LTQ where MSn analysis can be performed, or the ions are passed on to the C-tr ap. Ions that are transmitted to the C-trap are collisionally cooled there before injection into the orbitrap for detection or into the HCD cell for fragmentation. The use of nitrogen at higher pressures in the HCD cell creates higher energy colli sions than those typi cally found in the lin ear ion trap, which leads to a greater number of fragment ions.138 The octapole geometry of the HCD cell also allows for a lower mass cut off, making detectio n of low-mass ions possible. Fragment ions produced by collision-induced disso ciation in the HCD cell are transf erred back to the C-trap and then injected into the orb itrap for detection. Sample Analysis MALDI-LTQ/Orbitrap Individual sample wells containing the lipid ex tract were interrogated by spiraling the laser beam out from the center of the wells for 50 la ser steps where areas of good co-crystallization were observed. Microscope slides affixed with coated tissue were adhered to a modified MALDI sample plate using copper tape (3M, St. Pa ul, MN), and the entire sample was analyzed at a laser raster size of 100 m. All experime nts were performed with automatic gain control turned (AGC) on, and the signal was optimized by manually adjusting the laser power. Optimum mass spectra were base d on a signal of at least 103 for the lipid base peak (m/z 782.5) with little to no mass shifting, which indicated no affects of space charging. Full scan MS experiments were typically r un at a laser power of 15 20 J. MS2 experiments required the same laser power, with AGC adjusting the number of laser shots per step and were performed in the LTQ due to extensive fragmentation of lipid ions that occurred in the HCD cell. Qualitative data were obtained using standard Xcalib ur software with orbitrap detection. 103

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Nanospray-LTQ/Orbitrap An aliquot of 10l of the lipid extract was injected into a nanospray tip and then inserted into the nanospray probe. The probe was operate d at 1.5 kV in the positive-ion mode as direct infusion. Experiments were performed with automa tic gain control turned on with an ion inject time into the LTQ of 3 ms. CID was performed in the LTQ for all MS/MS experiments. Qualitative data were obtained using standard Xcalibur software with orbitrap detection. LC-ESI-MS/MS Liquid chromatography was performed on a gradient HP1100 HPLC (Agilent Technologies, Santa Clara, CA) system using an aqueous mobile phase A (1% formic acid, 5 mM ammonium formate) and an organic mobile phase B (isopropanol, 1% formic acid). The mobile phase flow rate was 0.3 mL/min, which be gan at a composition of 50% A and B, and was linearly ramped to 80% B over 30 min. A P honosphere column (Phenomenex, Torrance, CA) with dimensions 5 cm x 4.6 mm and 5 m particle size was used. The mass spectrometer was a Thermo Finnigan triple quadrupole TSQ 7000 (T hermo, Waltham, MA,), operated in positive ion parent scan mode. The sample was introduced through an electrospray ionization source. The second quadrupole was used as a collision chamber with argon as a collision gas. Sphingomyelins (SPM), phosphatidylcholines (PC), and lysophosphatidylcholines were analyzed in the parent ion scan mode by monitori ng all parent ions that lost m/z of 184.1. Results and Discussion LC-ESI and Nanospray Studies initially investigated the utility of LC-ESI-MS/MS of a lipid extract of 120m sections of rat spinal cord. Figure 5-1A illustrates a chromatographic separation obtained for a 10l injection of the spinal cord extract with MS detection set to scan for the m/z 184 parent ion. The LC peaks at times 8.40, 10.35, 13.12, 14.87, 16.70, 18.38 minutes correspond to elution 104

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105 of the internal standa rd PC(12:0,12:0), two sets of SPMs, PC(32:0), and two sets of PCs, respectively. Figure 5-1B is a spectrum averaged over the SPM peaks from 9.5 to 14.2 minutes, and Figure 5-1C is a spectrum averaged over the PC peaks from 16.9 to 20.0 minutes. In both lipid classes, lipids elute in the order of increasing number of carbons in the fatty acid tails. The SPMs detected are (18:0), (20:0), (22:1), (22:0), (24:1), and (2 4:0) at m/z values 731, 759, 785, 787, 813, and 815, respectively; however, only four PCs were observed, which were (32:0), (34:1), (36:1), and (38:6) at m/z valu es 734, 760, 788, and 806, respectively. Possible odd chained PCs are observed at m/z values 744, 746, 772, and 774, but confirmation of this assignment was not possible. PCs with longer ch ain fatty acids were not observed due to the high aqueous content of the mobile phase. This methodology was repeated three times for three different sets of tissue, but lack of reproducib ility in chromatographic separation was observed (replicates not shown). The amount of tissue analyzed proved a large enough quantity for mass spectrometric detection, but was insufficient for reproducible chromatographic analyses. Direct infusion experiments were therefore investigated to address the disadvantages experienced with chromatography. Positive-ion analysis of extracted lipids produces mass spectra dominated by singly-charged, protonated species. Such a spectrum is illustrated in Figure 5-2. The dominant ions in the spectrum are identified as PCs and Cers; however, close examination of any number of the mass peaks rev eals that more than one species is present at any given nominal mass. The high resolving po wer and accurate mass measurement achieved by the

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106 Figure 5-1. Lipid extract by LC-ESI-MS/MS. A) Chromatographic separation of tissue ex tract with MS detection mode of m/z 184 parent scan. B) Mass spectra of sphi ngomyelins averaged over chromatogra phic peak at10.35 and 13.12 minutes. C) Mass spectra of phosphatidylcholines averaged over ch romatographic peaks at 16.70 and 18.38 minutes. Relative Abundance 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 0 50 100 8.40 16.70 18.38 13.12 10.35 14.87 730 750 770 790 810 m/z 0 50 100 760.8 761.8 788.8 762.8 746.9 786.8 763.7 790.8 744.9 806.8 772.9 774.8 782.8 Relative Abundance 720 740 760 780 800 820 m/z 0 50 100 813.9 759.9 815.9 787.9 761.8 785.9 817.8 789.9 731.7 811.6A BCRelative Abundance 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 0 50 100 8.40 16.70 18.38 13.12 10.35 14.87 Relative Abundance 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 0 50 100 8.40 16.70 18.38 13.12 10.35 14.87 730 750 770 790 810 m/z 0 50 100 760.8 761.8 788.8 762.8 746.9 786.8 763.7 790.8 744.9 806.8 772.9 774.8 782.8 730 750 770 790 810 m/z 0 50 100 760.8 761.8 788.8 762.8 746.9 786.8 763.7 790.8 744.9 806.8 772.9 774.8 782.8 Relative Abundance 720 740 760 780 800 820 m/z 0 50 100 813.9 759.9 815.9 787.9 761.8 785.9 817.8 789.9 731.7 811.6 Relative Abundance 720 740 760 780 800 820 m/z 0 50 100 813.9 759.9 815.9 787.9 761.8 785.9 817.8 789.9 731.7 811.6 720 740 760 780 800 820 m/z 0 50 100 813.9 759.9 815.9 787.9 761.8 785.9 817.8 789.9 731.7 811.6A BC

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107 Figure 5-2. Mass spectrum of lipids detected from tissue extract by di rect infusion nanospray ionization. Figure 5-2. Mass spectrum of lipids detected from tissue extract by di rect infusion nanospray ionization. 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 760.583 8 788.6146 813.6826 746.6046 701.4922 731.6050 734.5683 747.6080 786.5992 759.6362 774.6357 816.6459 762.5884 790.6205 810.6799 828.6906 772.6197 806.5676 844.6774 826.6748 872.7090 842.6613 834.5989 870.6927 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 788.6146 813.6826 746.6046 701.4922 731.6050 734.5683 747.6080 786.5992 759.6362 774.6357 816.6459 762.5884 790.6205 810.6799 828.6906 772.6197 806.5676 844.6774 826.6748 872.7090 842.6613 834.5989 870.6927 8 760.583 100Relative Abundance

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orbitrap aid in identification of these ions, but fatty acid assignment to the sn -1 and sn -2 carbons is not possible. Studies have shown that lipids tend to be frag ile ions in the gas phase, and MS/MS of protonated species exhi bits a high degree of fragmentat ion to produce ions from the head group and little in the way of ions representative of the fatty acid tails.18 Table 5-1 lists the mass peak assignments for both high and low abundance ions for each ionization method used in this study. In total, 30 species were identified at 22 nominal m/z values, which include 14 PCs, 3 SPMs, 3 PEs, and 10 Cers. The SPM content of nerve tissue has been shown to be significantly less than that of PCs, and is represented accordingly in the presented data; however, the content of PEs has been reported to be equal to or greater than PCs.12 Previous work has demonstrated that PEs are more fragile in ion trap detection than PCs or SPMs,18 which may explain the apparent low abundance presented in the data here. Cers are presen t in a relatively high abundance in myelinated tissue and contain long-c hain fatty acids which are more likely to be odd-numbered when compared to other lipid classes.12 No ions were detected as PSs, suggesting that this lipid class experiences a higher degree of fragility and is not detectable in the protonated form.139 The addition of small amounts of alkali metals (nmol/ l) to the solution may increase the gas phase stability of these i ons and allow detection but may al so cause interferences with the ionization process. MALDI The detection of ions directly from nerve ti ssue with additional Na by MALDI (Figure 5-3) results in a different distributi on of lipid ions when compared to nanospray, as can be seen by comparing Figures 5-2 and 5-3. Because MALDI is much more tolerant to salt content, alkali metal cations may be added in abundance durin g matrix application. The addition of such cations has shown to reduce ion fragility and to produce highly informative fragmentation during collision-induced dissociation for lipids.140-142 The addition of catio ns contributes to the 108

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Table 5-1. Mass peak assignments for lipids detected us ing direct infusion nanospray (nESI) of lipid extracts, MALDI of lipid extracts (M ALDI-e), and direct analysis of tissue by MALDI (MALDI-t) m/z Peak assignment Molecular speciesDetection method 701.4922 PC(34:1) N(CH3)3[M+H]+nESI 723.4970 PC(16:0,18:1) N(CH3)3[M+Na]+MALDI-t,MALDI-e 731.6050 SPM(18:0) [M+H]+nESI 734.5683 PC(32:0) [M+H]+nESI 734.5721 PC(16:0,16:0) [M+H]+MALDI-e 739.4712 PC(16:0,18:1) N(CH3)3[M+K]+MALDI-t,MALDI-e 746.5696 PE(36:1) [M+H]+nESI 746.6046 Cer(d18:0,18:0h) [M+H]+nESI 750.5799 Cer(d18:1,18:0) [M+Na]+MALDI-e 751.5285 SPM(18:1) [M+Na]+MALDI-t,MALDI-e 753.6917 SPM(18:0) [M+Na]+MALDI-t,MALDI-e 754.5389 PC(34:4) [M+H]+MALDI-e 756.5553 PC(16:0,16:0) [M+Na]+MALDI-t,MALDI-e 759.6362 SPM(20:0) [M+H]+nESI 760.5838 PC(34:1) [M+H]+nESI 760.5889 PC(16:0,18:1) [M+K]+MALDI-t,MALDI-e 760.6387 Cer(d18:0,19:0h) [M+H]+nESI 767.5027 SPM(18:1) [M+K]+MALDI-t 768.5884 PE(18:0,18:1) [M+Na]+MALDI-t 769.5558 SPM(18:0) [M+K]+MALDI-t 769.6917 PC(16:0,22:6) N(CH3)3[M+Na]+MALDI-t 772.5838 PC(16:0,16:0) [M+K]+MALDI-t 774.5997 PE(38:1) [M+H]+nESI 774.6357 Cer(d18:0,20:0h) [M+H]+nESI 776.5965 PE(18:0,20:0) [M+K]+MALDI-t,MALDI-e 778.6111 Cer(d18:1,20:0) [M+Na]+MALDI-e 781.5905 SPM(20:0) [M+Na]+MALDI-t 782.5680 PC(36:4) [M+H]+nESI 782.5706 PC(16:0,18:1) [M+Na]+MALDI-t,MALDI-e 786.5992 PC(36:2) [M+H]+nESI 788.5210 PE(18:1,20:4) [M+Na]+MALDI-t,MALDI-e 788.6146 PC(36:1) [M+H]+nESI 788.6199 PC(18:0,18:1) [M+K]+MALDI-t,MALDI-e 109

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Table 5-1. Continued m/z Peak assignment Molecular speciesDetection method 798.5448 PC(16:0,18:1) [M+K]+MALDI-t 802.5583 PE(40:1) [M+H]+nESI 802.6663 Cer(d18:0,22:0h) [M+H]+nESI 806.5116 PE(18:0,20:4) [M+K]+MALDI-t 806.5676 PC(38:6) [M+H]+nESI 806.5722 PC(16:0,20:3) [M+Na]+MALDI-t,MALDI-e 806.6517 Cer(d18:1,22:0) [M+Na]+MALDI-t 808.5865 PC(18:1,18:1) [M+Na]+MALDI-t,MALDI-e 808.6675 Cer(d18:0,22:0) [M+Na]+MALDI-t,MALDI-e 810.5992 PC(38:4) [M+H]+nESI 810.6018 PC(18:0,18:1) [M+Na]+MALDI-t,MALDI-e 810.6799 Cer(d18:1,24:1) [M+H]+nESI 813.6826 SPM(24:1) [M+H]+nESI 816.5733 PC(38:1) [M+H]+nESI 816.6459 Cer(d18:0,23:0h) [M+H]+nESI 820.4379 PS(16:0,18:2) [M+Na+K]+MALDI-e 822.6464 Cer(d18:1,22:0h) [M+Na]+MALDI-t,MALDI-e 824.4641 PS(16:0,20:6) [M+2Na]+MALDI-t 824.5604 PC(18:1,18:1) [M+K]+MALDI-t 824.6173 PE(40:1) [M+Na]+MALDI-t 824.6621 Cer(d18:0,22:0h) [M+Na]+MALDI-t 826.5761 PC(18:0,18:1) [M+K]+MALDI-t 826.6748 Cer(d18:1,24:1h) [M+H]+nESI 828.5555 PC(16:0,22:6) [M+Na]+MALDI-t 828.5828 PE(18:1,22:6) [M+K]+MALDI-t 828.6906 Cer(d18:1,24:0h) [M+H]+nESI 832.5266 PS(18:1,18:1) [M+2Na]+MALDI-t,MALDI-e 832.5865 PC(18:0,20:4) [M+Na]+MALDI-t,MALDI-e 832.6671 Cer(d18:1,24:1) [M+Na]+MALDI-t,MALDI-e 834.5989 PC(40:6) [M+H]+nESI 834.6824 Cer(d18:1,24:0) [M+Na]+MALDI-t,MALDI-e 835.6702 SPM(24:1) [M+Na]+MALDI-t,MALDI-e 836.6174 PC(18:0,20:2) [M+Na]+MALDI-t,MALDI-e 836.6729 Cer(d18:1,23:0h) [M+Na]+MALDI-t,MALDI-e 110

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111m/z Peak assignment Molecular speciesDetection method 836.6975 Cer(d18:0,24:0) [M+Na]+MALDI-t,MALDI-e 837.6851 SPM(24:0) [M+Na]+MALDI-t,MALDI-e 838.6209 Cer(d18:0,23:0h) [M+Na]+MALDI-t 842.6613 PC(40:2) [M+H]+nESI 844.4694 PS(16:0,20:4) [M+Na+K]+MALDI-t 844.5293 PC(16:0,22:6) [M+K]+MALDI-t 844.6774 PC(40:1) [M+H]+nESI 848.5603 PC(18:0,20:4) [M+K]+MALDI-t+Table 5-1. Continued 848.6417 Cer(d18:1,24:1) [M+K] MALDI-t 848.6617 Cer(d18:1,24:1h) [M+Na]+MALDI-t 848.6975 Cer(d18:1,25:0) [M+Na]+MALDI-t 850.5010 PS(18:0,18:1) [M+Na+K]+MALDI-t 850.6578 PC(38:3) [M+K]+MALDI-t 850.6770 Cer(d18:1,24:0h) [M+Na]+MALDI-t,MALDI-e 851.6805 SPM(24:1) [M+K]+MALDI-t 856.4663 PS(18:0,20:4) [M+2Na]+MALDI-t 856.5265 PE(42:7) [M+K]+MALDI-t 856.5863 PC(18:0,22:6) [M+Na]+MALDI-t 864.6361 Cer(d18:1,24:1h) [M+K]+MALDI-t 864.6930 Cer(d18:1,25:0h) [M+Na]+MALDI-t 866.6514 Cer(d18:1,24:0h) [M+K]+MALDI-t,MALDI-e 870.6927 PC(42:2) [M+H]+nESI 872.7090 PC(42:1) [M+H]+nESI 874.5809 PC(40:5) [M+K]+MALDI-e 875.7340 PC(16:0,18:1) N(CH3)3 + (DHB H2O) [M+K]+MALDI-e 879.7442 SPM(26:1) [M+K]+MALDI-e 881.7600 SPM(26:0) [M+K]+MALDI-e

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112 Figure 5-3. Mass spectrum of lipids dete cted directly from tissue by MALDI Figure 5-3. Mass spectrum of lipids dete cted directly from tissue by MALDI 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 782.570 6 798.5448 810.6018 850.6770 723.4970 783.5741 739.4712 832.6671 776.5965 826.5761 835.6702 751.5285 866.6514 848.6617 822.6464 753.5917 767.5027 756.5553 808.5865 864.6361 760.5889 709.5176 838.6209 784.5772 792.5704 750.5809 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 782.570 6 798.5448 810.6018 850.6770 723.4970 783.5741 739.4712 832.6671 776.5965 826.5761 835.6702 751.5285 866.6514 848.6617 822.6464 753.5917 767.5027 756.5553 808.5865 864.6361 760.5889 709.5176 838.6209 784.5772 792.5704 750.5809

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complexity of the spectrum by presenting [M+H]+, [M+Na]+, and [M+K]+ as detectable ions. Similar to nanospray of extracts, the mass spectrum directly from tissue is dominated by PCs and SPMs which is due, in part, to decreased ion fragility and increased ionization efficiency when measured against other lipid species.96,143 However, a benefit of using IP MALDI is a reduction of in-source fragmentation cause d by collisional coo ling, allowing the detection of relatively fragile ions. A total of 59 ions were identified at 36 diffe rent nominal m/z values for lipids detected by MALDI directly from tissue. This includes 22 PCs, 8 SPMs, 17 Cers, 6 PEs, and 6 PSs. Accurate mass measurements and MS/MS of catio nized species allowed the assignment of the fatty acid composition for most ions. Dete rmination of fatty acid composition becomes increasingly difficult as the number of carbons in the fatty acid tails equal 20 or more. The majority of ions detected below m/z 800 were id entified as individual molecular species. While multiple peaks were observed at these nominal m/z values, they were identified as contributions from isotopic distributio n of other ions. In contrast, almost every nominal m/z value over 800 amu consists of at least two different species which are dominant molecular ions. Most of the ions identified by nanospray of extracts were also observed in the MALDI spectrum directly from tissue. However, 8 i ons were present exclus ively in the nanospray spectrum. They include 2 cerebrosides [(d18:0,1 8:0h), (d18:0,19:0h)], 1 PE [(38:1)], and 5 PCs [(36:4), (38:1), (40:1), (42:2), (4 2:1)]. The Cers detected in the direct MALDI experiment of direct tissue consist of fatty acid s that are 22 or more carbons in length. The majority of PEs seen in the same experiment has fatty acid tails with high degrees of unsaturation. In general, the PCs observed are composed of fatty acids with relatively low degrees of unsaturation. The differences noted in these ions suggest dissimilar ionization effi ciencies not onl y between lipid 113

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114 classes but among fatty acid composition within a speci fic class as well. A total of 19 lipid ions were identified in the MALDI spectrum directly from tissue that was not identified in the nanospray spectrum of extracts. They include 6 Cers [(d18:1,22:0), (d18:0,22:0), (d18:1,22:0h), (d18:1,24:0), (d18:1,25:0), (d18:1,2 5:0h)], 1 SPM [(18:1)], 5 PEs [(18:0,20:0), (18:1,20:4), (18:0,20:4), (18:1,22:6), (42:7)], 2 PCs [(16:0,20:3), (38:3)], a nd 5 PSs [(16:0,20:6), (16:0,20:4), (18:1,18:1), (18:0, 18: 1), (18:0,20:4)]. The analysis of lipid extracts with additional Na by MALDI can be seen in Figure 5-4. The distribution of ions resemble s a combination of the spectra obt ained directly from tissue and nanospray. The spectrum exhibits ions corre sponding to the protona ted molecular species predominately in the m/z region below 800 amu. Cationized lipids are distributed throughout the entire mass range that was analyzed. As in the previous two experiments, the lipid PC(16:0,18:1) is the most abundant and is obs erved here in the three forms [M+H-N(CH3)3]+, [M+H]+, and [M+Na]+ at m/z values 701, 760, and 782, respec tively. The relative abundance of m/z 782 is equal to that seen in the MALDI spectrum taken directly from tissue. However, other ions corresponding to [M+Na]+ species exhibit a decrease in re lative abundance. This is also noted for ions that are in the potassiated form as well, specif ically m/z 798, which is the second most abundant ion when analyzed by MALDI dire ctly from tissue, and is <1% in the MALDI spectrum taken from the extract. This is most likely due to the removal of cations during the extraction procedure, and while sodium is re introduced in the MALD I matrix, no additional potassium ions are added. The ion at m/z 760 in the MALDI extract spectrum is higher in abundance than the ion observed in the direct tissue analysis but less than the ion in the nanospray experiment. Also, the ion at m/z 701 produced from MALDI of th e extract is greater than that produced by the nanospray analysis of the extract. The relative abundance of these ions

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115 Figure 5-4. Mass spectrum of lipids de tected from tissue extract by MALDI 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 782.5701 701.4963 760.5881 723.4963 727.0264 820.4379 875.7340 810.6011 835.6693 756.5542 776.5957 850.6770 778.6111 837.6846 788.6193 754.5389 750.5799 832.6665 739.4062 822.6458 705.0446 879.7442 734.5721 874.5809 718.4659 866.6637 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 760.5881 723.4963 727.0264 820.4379 875.7340 810.6011 835.6693 756.5542 776.5957 850.6770 778.6111 837.6846 788.6193 754.5389 750.5799 832.6665 739.4062 822.6458 705.0446 879.7442 734.5721 874.5809 718.4659 866.6637 782.5701 701.4963 100Relative Abundance

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demonstrates that protonated li pid ions are more fragile when formed by MALDI compared to nanospray. Another notable difference in the MALDI spectrum of the ex tract in Figure 5-4 is the detection of ions in the mass range above 870 amu, which are not detected in the nanospray analysis of the extract or th e direct tissue analysis by MALD I. The ions at m/z 874, 875, 879, and 881 can be identified as [PC(40:5)+K]+, [PC(16:0,18:1)+K-N(CH3)3+(DHB-H2O)]+, [SPM(26:1)+K]+, and [SPM(26:0)+K]+, respectively. As stated earlier, other potassiated species exhibit a decrease in relative abundance in the MALDI spectrum of the extract, while the ions discussed here show a similar abundance to other abundant lipids in this mass range. Three of these four ions are composed of long chain fatty acid tails; the ion at m/z 875 is the only lipid detected as a MALDI matrix cluster. This may su ggest that larger, higher molecular weight lipid ions are more stable with the addition of al kali metals that are heavier than sodium. Conclusions In this work, three different ionization methods were approached for the analysis of lipids in rat nerve tissue. Extracted lipids were analyzed by both direct infusion nanospray and MALDI, and the direct interroga tion of intact tissue was perf ormed using MALDI. A hybrid LTQ/orbitrap mass spectrometer was used for mass analysis. The linear ion trap provided MSn capabilities while the orbitrap allowed fo r high resolution spectra and accurate mass determinations. Nanospray experiments produced protonated ions that were identified as 30 different lipid species, which were predominatel y PCs and Cers. SPM and PE ions were also detected, but PS ions were too fr agile in the protonated form to allow detection. MALDI of intact tissue with additional Na re sulted in the formation of [M+H]+, [M+Na]+, and [M+K]+ ions. A total of 59 molecular species were identified and included Cers, SPMS, PCs, PEs, and PSs. All PS ions were detected as double cationized species. Lipid extracts analyzed by MALDI 116

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resembled a combination of the data obtained in the other two experiments. However, 1 ion was identified as a lipid-matrix clus ter and 3 ions were found to cons ist of long chain fatty acids, which may suggest that MALDI of lipid extracts is more amenab le to the detection of higher molecular weight lipids. A comparison of the three ionization techniqu es used here also demonstrated that lipid ions tend to more fragile when produce by MALDI compared to nanospray. The direct analysis of tissue proved to be more sensitive than the tissue extract experiments. Analysis was accomplished with the use of one 10m section, which is at least an order of magnitude smaller than the sample si ze required for tissue extr action. This technique also affords the opportunity to provide localiza tion of lipids within tis sue. However, when employed in combination with one another, the techniques applied in this study give a more comprehensive understanding to the complex investigation of li pids in tissue. 117

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CHAPTER 6 CONCLUSIONS AND FUTURE WORK The goal of this research was to characterize the lipid content of nerve tissue by employing the technique of mass spectrometric imaging (MSI). Lipids play a critical role in the structure and function of the nervous system, and MSI allows the analysis of these vital compounds to be investigated for both chemical structure and spatial distribution within tissue. This 2dimensional representation may provide insight into the chemically specific profile of lipids in tissue, how lipids are affected by an exogenous compound, and how lipids affect or are effected by a diseased state. Initial studies were able to identify rela tively abundant lipid ions based on MS/MS analyses at unit resolution. Low abundance and/ or isobaric species were identified using MSn. In positive ion mode, a total of five lipid classe s were identified in nerve tissue, which included phosphatidylcholines (PC), phosphatidylethanol amines (PE), phosphatidylserines (PS), sphingomeylins (SPM), and cerebrosi des (Cer). PCs, PEs, SPMs, and Cers were detected as several molecular speci es including [M+H]+, [M+Na]+, and [M+K]+. PSs were detected as doubly cationized species such as [M+2Na]+ or [M+Na+K]+, and often produced the most informative fragmentation for id entification purposes. Gas-phas e stability of doubly cationized lipids was not investigated here but a possible route for future study is using both MALDI and electrospray ionization of standards to compare th e fragility of these ions to other lipid ions during transport and analysis in a quadrupole ion trap. The tissue rastering capabilities of the MSI technique were employed to semiquantitatively compare the lipid content of nerve tissue taken fr om control animals and animals that had been administered dichloroacetate (DCA ), a possible neurotoxi n. Signal from 8 lipids was averaged over an entire tissue section from both types of specimens, and an average 118

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decrease of about 4 fold for each lipid was observed in the DCA-administered tissue when compared to control tissue. The precisi on for these measurements was within 20% SE. Further quantitative measures were first expl ored by monitoring successive analyses of a single tissue section to provide insight into the variation of signal respon se with an increasing number of laser shots. Such an experiment was necessary to minimize errors due to signal variability and for direct comparisons to other MS and quantitative techniques. Therefore, all comparative studies were conducted by analyzing tissue sections multiple times and averaging the signal response. Another effort employed to reduce signal variation and provide a means of more traditional quantitation was the application of an internal standard to the tissue. A non-endogenous phospholipid, PC(10:0,10:0), was explored for use as an internal standard. It was chosen because it does not interfere with ions typically found in the lipid mass range (m/z 700 900) as well as having similar ionization an d extraction efficiency to the endogenous lipids of interest. Three different application methods of the inte rnal standard were ev aluated: 1.on top of dry tissue, 2. on top of wet tissue, 3. under wet tissue. For quantitative purposes, it was determined that application of the internal standard on top of dry tissue pr ovided the most even coating and signal distribution. However, generation of tissu e images showed that the greatest spatial resolution and most accurate anatomical struct ure are observed by applying internal standard under wet tissue, which may be due to analyte migr ation during the wetting of the sample surface during the application of the internal standard. Endogenous PCs were quantified in brain and spin al cord tissue sections by a ratio of the analyte signal response to the internal standard signal response. Results showed that quantitation performed by MALDI was similar when compared to published results. The average precision 119

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for measurements taken from spinal cord sec tions was 4%, which is quite remarkable for MALDI analysis. The brain section was only an alyzed once, so no precision was able to be calculated; however, quantitative results still exhibited a marked similarity to literature values. More reliable quantitation may be achieved for future studies by generating a calibration curve measuring the response of differing concentrations of internal standard applied to tissue. An attempt to quantitate other lipid cla sses found in nerve tissue presented a unique challenge. The preformed positive charge of the quaternary amine of PCs and SPMs enhances the detectability of these lipids over that of other lipid species. Therefore, a more reliable method for quantifying low abundance ions would be through MS/MS analyses. Due to the presence of isobars, the quantitation of low abundance species could be accomplished through MS/MS experiments; however, a separate MS/MS s can would have to be performed for the internal standard which would in troduce greater uncertainty in the quantitation process. An alternative and promising techniqu e for the quantitation of isobaric species is the analysis of tissue using high mass resolution. Multiple internal standards could be applied to tissue, and one full scan would produce data for each internal st andard and all the isoba rs of interest. Full-scan mass spectra and ma ss spectral images were comp ared between data acquired on an orbitrap and a LIT. The increased mass spectral resolution on the orbitrap provided mass spectra with substantially le ss chemical background. The reduction in background made observation of variations in composition in different tissue regions more easily distinguishable. Background reduction was also clearly evident in mass spectrometric images, which enabled a more accurate visualization of the anatomical di stribution of lipids. Further studies in which only the MALDI matrix is ionized could be inve stigated to provide more insight into the background reduction observed in these experiments. 120

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A difference in the ratio of [M+H]+ and [M+K]+ ions was also observed in the spectra produced from the two mass analyzers. When compared to the LTQ spectra, the relative abundance of [M+H]+ ions was decreased and the relative abundance of [M+K]+ ions was increased in the orbitrap spectra. Because of the increased gas-pha se stability observed for alkali metal adducts, it is hypothesized th at the increased storage time of orbitrap analysis causes greater fragmentation of the less stable [M+H]+ ions. Experiments designed to test this hypothesis may include increasing the storage time of ions in the linear ion trap. A comparison of MS images was also made between data produced from each mass analyzer. The images generated from the LIT encompassed a 1 amu wide window corresponding to unit mass resolution. This method gives an accurate representation of the ion distribution of the most intens e ion within the 1 amu window. Analysis of the same 1 amu window by the orbitrap with mass resolution of 100,000 showed several ions each baselineresolved with a mass accuracy of typically ~3 ppm. Images of each ion generated with a 0.02 amu window showed diverse distributions within the tissue sample, which proved beneficial for identification. Identification of ions was achie ved by employing both mass analyzers. Orbitrap, full-scan HRMS images were matched to LTQ /orbitrap MS/HRMS images. Assignment of daughter ion accurate mass and neutral lo sses allowed for rapid identification. The MSn and high resolution cap abilities of the hybrid linear i on trap/orbitrap were utilized for lipid identification in nerve tissue employing three different ionization methods. Extracted lipids were analyzed by both direct infusion na nospray and MALDI, and the direct interrogation of intact tissue was performed using MALDI Nanospray experiments produced protonated ions that were identified as 30 different lipid species, which were predominately PCs and Cers. SPM and PE ions were also detected, but PS 121

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ions were too fragile in the protonated form to allow detection. MALDI of intact tissue with additional Na resulted in the formation of [M+H]+, [M+Na]+, and [M+K]+ ions. A total of 59 molecular species were identified and included Cers, SPMS, PCs, PEs, and PSs. All PS ions were detected as double cationized species. Li pid extracts analyzed by MALDI resembled a combination of the data obtained in the other two experiments. However, 1 ion was identified as a lipid-matrix cluster and 3 ions were found to consist of long chain fatty acids as potassium adducts, which may suggest that MALDI of lipid extracts is more amenable to the detection of higher molecular weight lipids as adducts of large alkali meta l ions. Future experiments investigating the preferential i onization by alkali metal cations could be determined by the addition of ions heavier than potassium. A comp arison of the three ionization techniques used here also demonstrated that lipid ions tend to more fragile when produce by MALDI compared to nanospray. Direct analysis of tissue prove d more sensitive than tissue extract experiments. Analysis was accomplished with the use of one 10m section, which is at least an order of magnitude smaller than the sample size required for tissue extraction. This technique also affords the opportunity to provide localizat ion of lipids within tissue. However, when employed in combination with one another, the techniques ap plied in this study give a more comprehensive understanding to the complex investigation of lipids in tissue. 122

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BIOGRAPHICAL SKETCH Rachelle Renee Landgraf was born in 1980, the fi fth of seven children to Fred and Joyce Biery. She grew up in the small town of Aust well, Texas, where she graduated from AustwellTivoli High School with nine other people in May 1999. In August 1999, she married her high school sweetheart, Jeffery. Rachelle received her bachelor's degree in chemistry from Texas A&M UniversityCorpus Christi in May 2004. As an undergraduat e, she worked under the advisement of Eugene and Feri Billiot, synthesizing ch iral surfactants for the separati on of chiral drug compounds as well as characterizing endocrine disrupting compounds (EDCs) in environmental samples using gas chromatography/mass spectrometry. When not conducting research, she spent most of her time on Padre Island National Seashore shark fishing with her husband. Rachelle began her graduate career at th e University of Florida in August 2004 under the advisement of Richard A. Yost. Her graduate research started by usi ng high-field asymmetricwaveform ion mobility spectrometry (FAIMS) to analyze steroids, proteins, and EDCs. She eventually switched the focus of her research to analyzing the lipid conten t of nerve tissue using mass spectrometric imaging. While a graduate st udent, she found her love for cake decorating. At completing her Ph.D., she joined her husband in Stuart, FL, where they still enjoy shark fishing. 135