UC San Diego UC San Diego Electronic Theses and Dissertations Title Novel proteomics methods for increased sensitivity, greater proteome coverage, and global profiling of endogenous SUMO modification sites Permalink https://escholarship.org/uc/item/4xq3v9wd Author Meyer, Jesse Gerard Publication Date 2015 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Novel proteomics methods for increased sensitivity, greater proteome coverage, and global profiling of endogenous SUMO modification sites A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry by Jesse Gerard Meyer Committee in charge: Professor Elizabeth A. Komives, Chair Professor Nuno Bandeira, Co-Chair Professor Jack Dixon Professor Randy Hampton Professor Judy Kim Professor Wei Wang 2015 Copyright Jesse Gerard Meyer, 2015 All rights reserved The dissertation of Jesse Gerard Meyer is approved, and it is acceptable in quality and form for publication on microfilm: Co-Chair Chair University of California, San Diego 2015 iii DEDICATION I dedicate this work to my family and friends. iv TABLE OF CONTENTS Signature Page…….……..………………………………………………………… iii Dedication……………………………………………………..…………………… iv Table of Contents………………………………………………………………... …v List of Abbreviations ……………………………………………………………… xi Lists of Figures……………………………………………….………….………… xiv Lists of Tables…………………………………………………...…….…………… xvii Acknowledgements………………………………………………………………… xviii Vita……………………………………………………………..……...…………… xx Abstract of the Dissertation……………………………………..…………………. xxi Chapter I Introduction…………………………………………….…………. 1 A. Bottom-up proteomics………...….………………….…….………. 2 C. Peptide electrospray and fragmentation..………..…………….…… 5 B. Proteome digestion………………..………………………….……..8 D. References………………………………………………………….. 11 Chapter II Charge State Coalescence During Electrospray Ionization Improves Peptide Identification by Tandem Mass Spectrometry ……………..…………………………………………..…………... 13 A. Introduction………….………....…………………..…………….… 14 B. Materials and methods………....…………………..…………….… 18 v 1. Samples and solutions…...………..……………..…………….. 18 2. Liquid Chromatography…………………...………...….....…… 18 3. nLC-MS/MS…………………….....……...…..…..…………… 19 4. Data Analysis……………...…….…………..………………… 20 C. Results……………………………………………..…………...….. 21 1. Co-solvent effects on the number of high quality peptide identifications ……………………………………………....... 21 2. Effects of co-solvents on chromatography …………….……... 22 3. Tandem mass spectrum quality ………………………..……… 28 4. Effects on peptide charge state …………………………..……. 32 5. Conclusions…….………………………...…………………….. 37 D. References………………………………………………………….. 40 Chapter III Expanding proteome coverage with orthogonal-specificity alpha- lytic proteases……………………………………………………... 44 A. Introduction…….……………………………………………..…… 45 B. Materials and methods…………..……………………………...….. 49 1. Samples and Chemicals ..…………...…………….……....…... 49 2. Protease expression and purification .….…..…..…………..…. 49 3. Protease activity assays ….………..………………….……..… 50 4. In gel digestion ………...………….…...……………………… 50 5. Proteome preparation and digestion.…...……………………… 51 vi 6. MS activation comparisons ……….…...……………………… 52 7. High-pH fractionation of proteome samples ………………… 53 8. NanoLC-ESI-MS/MS of HPRP-fractionated digests ………… 54 9. Database searches …………………………..………………… 54 10. Data Analysis…... …………………………..………………… 57 C. Results………..………………………………………..………..….. 58 1. Protease activity in SDC, SDS, and GdnCl..…...……...…….... 58 2. Coverage of standard protein mixture ………………...………..60 3. Comparison of tryptic and non-tryptic peptide identification using CID, ETD, and HCD ………………………………………...… 60 4. Characterization of the MSMS data from WaLP and MaLP digests………………………………………….….…….…….. 68 5. Substrate specificity of WaLP and MaLP …..………...……….. 71 6. Length of peptides from WaLP and MaLP digestions ..………..74 7. Quantitation of peptide overlap ……..………………...………. 78 8. Biological gains from WaLP and MaLP digestions…...………. 81 9. Discussion…………………… ……..………………...……….. 85 D. References………………………………………………………….. 89 Chapter IV In silico Proteome Cleavage Reveals Iterative Digestion Strategy for High Sequence Coverage ……………………….…..………... 94 A. Introduction………………………………………………….….….. 95 vii B. Materials and methods…………..……………………………..….. 98 1. In silico proteome digestion …………….….………………….. 98 C. Results….…………………..…………………..…..……………… 98 1. Minimum unique peptide length..……………………………… 98 2. Peptide length distributions from various cleavages………....... 99 3. Comparison of peptide filtration parameters .....……………..... 103 4. Comparison of digestion iterations …………………………..... 103 5. Proposed iterative digestion strategy ………………………..... 106 6. Conclusions …………………………………………………..... 106 D. References………………………………………………………….. 109 Chapter V Global, site-specific identification and quantitation of endogenous human SUMO modifications...……………………….………..… 113 A. Introduction………………………...…...………………………….. 114 B. Materials and Methods…………………..……..………………... 117 1. Cell culture and treatment ……….....…..……………………… 117 2. Cell lysis and digestion ……………….…...…………………... 118 3. Western blotting………...…………………...……………..…... 118 4. Off-line separation of peptides prior to IP ...…...……………… 119 5. nLC-MS/MS …………….…………………………………….. 120 6. Data Analysis ………………………...…………...………….... 120 C. Results………...……………...……………………..…...…………. 122 viii 1. Wild-type alpha-lytic protease (WaLP) digestion allows endogenous SUMO profiling………………………………………. 122 2. Global SUMO profile results……………………….…………. 123 3. SILAC quantitation of SUMO site changes in MG132 treated cells……………………………………………………………….... 127 4. Validation of biological results …………………….………….. 131 5. Discussion…………………..……………………….…………. 131 D. References..…...……………...……………………..…...…………. 136 Appendix A SUMO-remnant Data Analysis……………………….………..… 139 A. Database Searching...………...……………………..…...…………. 140 1. Convert .RAW files to mzXML files using TPP………………. 140 2. Database searching data from WaLP digestion using MSGF+... 140 3. Database searching tryptic data………………………………... 141 4. Fix the formatting in your output files from the heavy database search……………………………………………………………..... 142 5. Convert each .mzid files to .pep.xml…………………………... 145 B. TPP refinement of identifications…………………..…...…………. 146 1. Run Peptide Prophet…………………………...………………. 146 2. Run PTM Prophet…………………………………………….... 148 3. Combine the heavy and light database search output files .…... 150 4. Fix your PTM Prophet files for SILAC quantification.……..... 150 5. Filer by minimum iProphet score to apply 1% FDR…………... 151 ix 6. Filter the excel file to include only dglycyl-lysine peptides.…... 151 C. Post-identification processing…..…………………..…...…………. 155 1. Normalize distribution of quantification values, compute weighted average quantification values, and filter by localization score…………………………...………………………………….... 155 2. Compare SUMO-site identifications with previously reported modifications………..……………………………………………....184 3. Extract the sequence windows for motif analysis….................. 207 x LIST OF ABBREVIATIONS ACN Acetonitrile AGC Automatic Gain Control BCA Bicinchoninic Acid Assay BEH Bridged-Ethylene Hybrid BSA Bovine Serum Albumin CID Collision-induced Dissociation ddDT Data-dependent Decision Tree DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid EDTA Ethylene-diamine Tertiary Acetic Acid EIC Extracted Ion Chromatogram ESI Electrospray Ionization ETD Electron-transfer Dissociation FA Formic Acid FDR False-discovery Rate FPLC Fast Protein Liquid Chromatography FT Fourier Transform FWHM Full Width at Half Maximum HCD Higher-energy Collisional Dissociation HPRP High-pH Reversed Phase IAA Iodoacetamide xi LC Liquid Chromatography LC/MS Liquid Chromatography-Mass Spectrometry LTQ Linear Trap Quadrupole m-NBA meta-Nitrobenzyl Alcohol MaLP M190A Alpha-lytic Protease mRNA Messenger Ribonucleic Acid MS Mass Spectrometry MS/MS Tandem Mass Spectrometry MudPIT Multi-dimensional Protein Identification Technology NEM N-ethyl Maleimide NRAAS Non-redundant Amino Acid Sequences PAGE Poly-acrylimide Gel Electrophoresis PTM Post-translational Modification RNA Ribonucleic Acid SDC Sodium Deoxycholate SDS Sodium Dodecyl Sulfate SL Sodium Laurate SUMO Small Ubiquitin-like Modifier protein TFA Trifluoroacetic Acid TCEP Tris(2-carboxyethyl)phosphine TIC Total-ion Chromatogram TOF Time of Flight xii TPP Trans-Proteomic Pipeline WaLP Wild-type Alpha-lytic Protease xiii LIST OF FIGURES Figure 1.1. The bottom-up proteomics workflow ………………….………..… 3 Figure 1.2. A table describing the ion activation methods CID, HCD, and ETD ………………………………………………………….…..…...…. 7 Figure 1.3. A plot comparing the theoretical upper limits of proteome coverage with observed coverage ………………………………...…………..9 Figure 2.1. Comparison of TICs observed for each mobile phase condition …. 24 Figure 2.2. Extracted ion chromatograms (EICs) for the +1, +2, and +3 charge states of the elastic peptide, TEDELQDKIHPF …………………. 25 Figure 2.3. Global effects of supercharging reagents DMSO and m-NBA on chromatographic peptide retention during reversed-phase nano LC- ESI-MS/MS..…...………………………………………………..… 27 Figure 2.4. MS/MS spectra produced from the fragmentation of the doubly and triply charged precursor for the elastic peptide TEDELQDKIHPF .………………………………………………... 29 Figure 2.5. Comparison of unique peptide counts for each protease and each mobile phase condition …………………………………………..... 31 Figure 2.6. MS/MS spectra produced from