A novel mass spectrometry-based proteomic and phosphoproteomic strategy for the quantitative analysis of a leukemic cell line infected by an oncolytic virus by Matthew Huebsch A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Carleton University Ottawa, Ontario ©2011, Matthew Huebsch Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-81700-1 Our file Notre reference ISBN: 978-0-494-81700-1 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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To succeed as an experimentalist,you can go through life kicking over a lot of stones, and if you 're lucky, you '11 find something." -John B. Fenn 11 Abstract Mass spectrometry has become the tool of choice for large proteomic experiments to investigate the molecular dynamics of biological systems. A strategy for the proteomic and phosphoproteomic quantitative analysis of samples in different biological states has been developed. The strategy incorporates tryptic digestion of cellular lysates, on-column stable-isotope dimethyl labeling, and proteomic and phosphoproteomic investigations by SAX/SCX-LC-MS/MS and IMAC- LC-MS/MS, respectively. The strategy was applied to study altered protein expression and signaling pathways induced by infection of a leukemic cell line, K562, with the oncolytic vesicular stomatitis virus for 30 minutes. A total of 53 proteins and 8 phosphorylation sites were found to be upregulated and 11 proteins and 9 phosphorylation sites were found to be downregulated in VSV-infected K562 cells versus controls. Possible roles of differentially regulated proteins and phosphorylation sites in VSV infection are discussed and the performance of the developed quantitative strategy is evaluated. in Acknowledgments I'd like to thank my supervisor Jeff Smith for his guidance and patience throughout my time as a graduate student, as well as all other members of the group who have provided me with helpful feedback. I'd like to thank my collaborator Harry Atkins for ideas and resources and Girija Waghray of the Atkins laboratory for culturing and infecting the cells used in this work. I'd also like to extend my gratitude to Michel Dumontier for writing the script used in this work for the analysis of large datasets; without his insight and dedication, this work would not have been possible. An additional thanks to all professors and fellow students not mentioned herein who have enriched my experience as a graduate student. I'd also like to thank friends and family for always providing me with support. Thanks to Tim for always being a great brother and to my parents for their unconditional support and understanding. A specific thanks to Chris Bura for not only being a close friend, but for his willingness to proofread, give technical advice, and provide useful scripts. Finally, I'd like to thank Jinal Patel for encouragement and insight; you have been a good colleague and great friend. IV Table of Contents Abstract iii Acknowledgment iv Table of Contents v List of Tables viii List of Illustrations ix List of Appendices xii List of Abbreviations xiii 1.0 Introduction 1 1.1 Protein biochemistry 3 1.1.1 Post-translational modifications 9 1.2 Mass spectrometry 11 1.2.1 Electrospray ionization 14 1.2.2 Quadrupole mass analyzers 17 1.2.3 Time-of-flight 22 1.2.4 Ion detectors 27 1.2.5 Hybrid quadrupole-time-of-flight mass spectrometer 28 1.3 Mass spectrometry-based proteomics 30 1.3.1 Identification of peptides from spectra 34 1.3.2 Quantitation 36 1.3.3 Phosphoproteomics 40 v 1.4 Separation methods 43 1.5 Vesicular stomatitis virus 49 References 58 2.0 Experimental 67 References 75 3.0 Results and discussion 76 3.1 Analytical method development 76 3.1.1 Overview of the workflow 76 3.1.2 On-column versus in-solution digestion 77 3.1.3 Labeling strategies for peptide quantitation 80 3.1.4 Lysis conditions 85 3.1.5 Peptide fractionation 86 3.1.6 Peptide identification and quantitation 98 3.2 Qualitative K562 analysis 102 3.2.1 Background proteomic analysis 102 3.2.2 Background phosphoproteomic analysis 105 3.3 Quantitative VSV-infected K562 analysis 109 3.3.1 Quantitative proteomic analysis 109 3.3.2 Quantitative phosphoproteomic analysis 122 3.4 Conclusions 130 References 132 VI 4.0 Limitations and future directions 138 References 142 Appendices 143 vn List of Tables Table 1.1 Common post-translational modifications in proteins 10 Table 3.1 Examples of peptides showing partial methyl esterification and deamidation on glutamine and asparagine residues 83 Table 3.2 Classification of phosphopeptide missed cleavage sites 109 Table 3.3a Upregulated phosphopeptides in VSV-infected K562 124 Table 3.3b Downregulated phosphopeptides in VSV-infected K562 124 viii List of Illustrations Figure 1.1 The central dogma of molecular biology 2 Figure 1.2 The common 20 amino acids found in proteins 4 Figure 1.3 The four structural levels of proteins 7 Figure 1.4 Addition and removal of phosphates by kinases and phosphatases... 11 Figure 1.5 Typical mass spectrum 13 Figure 1.6 Electrospray ionization 15 Figure 1.7 Nanospray tips and setup 17 Figure 1.8 Quadrupole mass filter 18 Figure 1.9 Mechanism of quadrupole bandpass filtering 19 Figure 1.10 Quadrupole first stability diagram 21 Figure 1.11 Typical time-of-flight mass spectrometer 23 Figure 1.12 Reflectron time-of-flight mass spectrometer 24 Figure 1.13 Orthogonal acceleration region in TOF-MS 25 Figure 1.14 QqTOF mass spectrometer 30 Figure 1.15 Fragment ions formed in CID of peptides 33 Figure 1.16 Quantitation strategies in quantitative proteomics 39 Figure 1.17 Mechanism of phosphopeptide enrichment by Fe(III) IMAC 43 Figure 1.18 Polysulfoethyl aspartamide SCX resin 48 ix Figure 1.19 The structure of vesicular stomatitis virus 49 Figure 1.20 Pathways involved in VSV-induced apoptosis 55 Figure 2.1 Pressure vessel setup 71 Figure 3.1 Quantitative proteomics/phosphoproteomic strategy 77 Figure 3.2 Standard peptides identified after on-column or in-solution tryptic digestion 80 Scheme 3.1 Methyl esterification reaction for stable isotope labeling 81 Scheme 3.2 Dimethylation reaction for stable isotope labeling 83 Figure 3.3 Effect of dimethylation on SCX fractionation 88 Figure 3.4 Number of SCX and SAX fractions in which peptides appear 91 Figure 3.5 Overlap between identified peptides from SAX and SCX fractions 92 Figure 3.6 Number of phosphorylated vs. non-phosphorylated peptides in I MAC elution fractions 95 Figure 3.7 Overlap between identified phosphopeptides in two samples 97 Figure 3.8 MS/MS spectrum of phosphopeptide LELQGPRGpSPNAR with a Mascot score of 16 98 Figure 3.9 Manual XlC-based quantitation method 101 Figure 3.10 Functionally enriched GO terms in the background proteome 104 Figure 3.11 Phosphorylation motifs found in background phosphoproteome 107 Figure 3.12 Interaction network for proteins upregulated by VSV infection 113 x Figure 3.13 Interaction network for proteins with downregulated phosphorylation sites by VSV infection 125 Figure 3.14 Apoptotic pathways controlled by HDGF 128 XI List of Appendices Appendix 1 Exhaustive list of functionally enriched gene annotations in the background proteome 144 Appendix 2 Centered, 13-amino acid peptide sequences and their corresponding phosphorylation motifs 146 Appendix 3 Complete list of upregulated and downregulated proteins found in VSV-infected K562 cells relative to control K562 cells 147 Appendix 4 Exhaustive list of proteins identified in background proteome by gene name 150 XI1 List of Abbreviations ABC, ammonium bicarbonate BCA, bicinchoninic acid CID, collisionally induced dissociation ETD, electron transfer dissociation GO, gene ontology HDGF, hepatoma-derived