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Novel Biomedical Applications of Maldi and Electrospray Mass Spectrometry

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Novel Biomedical Applications of Maldi and Electrospray Mass Spectrometry NOVEL BIOMEDICAL APPLICATIONS OF MALDI AND ELECTROSPRAY MASS SPECTROMETRY by Kim Yoke Ching Fung A dissertation submitted in fulfillment of the requirements for the degree of Doctor of Philosophy University of New South Wales Sydney, Australia 2001 I hereby declare that this submission is my own work and to the best of my knowledge it contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged. December, 2001 ACKNOWLEDGEMENTS I would like to express my appreciation to the following people: • Prof. Mark Duncan for the support, advice and guidance with every aspect of my degree and for the many opportunities presented to me over the past four years. • Dr. George Smythe for being my co-advisor and for the support you have given me throughout my degree. • Dr. Carol Morris and Dr. Robert Sack for providing the tear samples and for your advice and insight regarding the data generated. • Dr. David Friedman for your help with the prostate fluid experiments. • Martin Bucknall for your help with the studies on quantitative MALDI. • Dr. Joseph Zirrolli for your day-to-day support, and for sharing your vast knowledge of mass spectrometry with me. • Staff and fellow students (past and present) from the labs I have interacted with during the course of my work for the great times experienced both in the lab and socially. Finally, I would like to thank my family and close friends for their continual love and support as well as for all the good times we have shared. ABSTRACT Over the last decade, developments in mass spectrometry have made the analysis of high molecular weight involatile biomolecules, including peptide and proteins, a reality. Concurrent expansions in protein and genomic databases, as well as progress in bioinformatics, have enabled proteins to be unambiguously identified using sensitive mass spectrometric techniques such as matrix assisted laser desorption ionisation (MALDI) and electrospray ionisation (ESI). This thesis investigates the application of mass spectrometry, in combination with techniques such as gel electrophoresis and liquid chromatography, for the analysis of peptides and proteins in biological fluids. Peptides and proteins in human tear fluid were characterised using a combination ofMALDI-TOFMS and ESI-LC/MS/MS. Direct analysis of tear fluid by MALDI-TOFMS detected over 30 peptides and proteins and MALDI-PSD identified homologous peptides derived from the C-terminus oflacrimal proline-rich protein. ESI-LC/MS/MS of tryptic peptides identified many more proteins, including known constituents of human tear and proteins previously not reported as components of the tear film. Seminal fluid proteins were separated by gel electrophoresis and identified by peptide mass fingerprinting. Although over 300 protein spots were detected, only 32 unique proteins were identified including semenogelin I, semenogelin II, prostate specific antigen and prostate secreted seminal plasma protein. The remaining proteins were identified as protein isoforms or modified proteins. Additional analysis by MALDI- TOFMS and ESI-LC/MS/MS identified many proteolytic products of semenogelin I and semenogelin II. The utility of mass spectrometry for the quantification of proteins and other biomolecules was also explored. The relative abundance of more than 20 proteins in open and closed eye tear fluid was measured using ESI-LC/MS following esterification of proteolytic peptides with isotopomers of butanol. Also, the potential ofMALDI-TOFMS for the absolute quantification of various biomolecules is explored. Rat growth hormone, human insulin, LW-hemorphin- 7, the catecholamines epinephrine and norepinephrine, and homovanillic acid were quantified in biological tissue or fluid using stable isotopomers or structural homologues as internal standards. The role of mass spectrometry in global protein identification and quantification is also discussed. TABLE OF CONTENTS LIST OF FIGURES ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• V LIST OF TABLES •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• VIII CHAPTER 1: INTRODUCTION 1 1.1. Introduction .•.••.•.••.•.••.•.••..•.•.•••.•...........•.........•...•.••.•.••.•..••.••.•.••.••..•.••.•.....••.••..•.••.••.. 2 1.2. Matrix assisted laser desorption ionisation ............................................................. 3 1.3. Electrospray mass spectrometry ............................................................................ 10 1.4. Ion dissociation and tandem mass spectrometry .................................................. 16 1.5. MALDI and ESI in the biological sciences ............................................................ 19 1.5.1. Quantification of biomolecules by mass spectrometry .......................................... 19 1.5.2. Analysis ofbiomolecules in complex mixtures ..................................................... 21 1.6. The emergence of Proteomics ................................................................................. 25 1.7. Aims ••••••••••••••••••••••••••••••••••••.•.••.•.••..•..•.•.••.••.••••.••.•.••.•.••.•.••.•.••..••••.••.•.•••••••••••••••••••.•• 27 CHAPTER 2: METHODS 31 2.1. Instrumentation················································~······················································ 32 2.2. MALDI matrices •.••••••••••••••••••••••••••••••••••••••••••••••••••••••••.•.•••••••••••.••••.•.••.•.••.•.•••••••••••• 33 2.3. Proteolytic en.zymes •.••••••.•••••.•.••.•.••.•.••.••.•.••.•.•.••...•••.••••••••••••••••.••••••••..••••.••••.•.••••••• 33 2.4. Protein identification criteria .••.•.••.•.••.••.•.•••••••••••••••.••.•.••.•..•.•.••.•..••••..•••.••••••••••••••• 34 2.4.1. MALDI-TOF mass spectrometry ........................................................................... 34 2.4.2. Electrospray mass spectrometry ............................................................................. 34 2.S. Database search criteria •.••..••.•.•••••••••••••....•.•.•••••••••••••••.••.•.••.•.••.•.••.•.••.•.••.•.••.•.•..•.• 35 2.6. Identification of protein constituents in human tear ........................................... 36 2.6.1. Sample collection ................................................................................................... 36 2.6.2. Analysis of whole tear sample................................................................................ 36 i 2.6.2.1. MALDI analysis .......................................................................................... 36 2.6.2.2. Electrospray mass spectrometry.................................................................. 37 2.6.3. Isolation and detection oflacrimal proline-rich protein ......................................... 37 2.6.4. Quantification of tear fluid proteins ....................................................................... 38 2.6.4.1. Apomyoglobin ............................................................................................ 38 2.6.4.2. Tear fluid proteins ....................................................................................... 39 2. 7. Analysis of the peptide and protein components in human seminal plasma ..... 41 2. 7 .1. Sample collection ................................................................................................... 41 2.7.2. Two dimensional gel electrophoresis ..................................................................... 41 2.7.3. One dimensional gel electrophoresis ...................................................................... 42 2.7.4. Mass spectrometry ofunfractionated seminal fluid ............................................... 43 2. 7 .5. Protein identification .............................................................................................. 43 2.7.5.1. MALDI-TOF mass spectrometry ................................................................ 43 2.7.5.2. ESI mass spectrometry ................................................................................ 44 2.8. Quantification of biomolecules by MALDI-TOFMS ........................................... 45 2.8.1. Analysis of growth hormone in rat pituitary tissue ................................................ 45 2.8.2. Quantification of insulin in human pancreatic tissue ............................................. 45 2.8.3. Analysis ofhemorphins in human adrenal and pheochromocytoma tissues .......... 46 2.8.4. Analysis of catecholamines in human adrenal and pheochromocytoma tissue ...... 47 2.8.5. Analysis ofhomovanillic acid in human urine samples ......................................... 48 2.8.6. Data acquisition and processing ............................................................................. 49 CHAPTER 3: MASS SPECTROMETRY AND PROTEIN IDENTIFICATION: APPLICATION TO HUMAN TEAR FLUID 50 3.1. Introduction ............................................................................................................
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