Proteomics of Biological Systems: Protein Phosphorylation Using Mass Spectrometry Techniques

PROTEOMICS OF BIOLOGICAL SYSTEMS: Protein Phosphorylation Using Mass Spectrometry Techniques......Page 5
CONTENTS......Page 9
PREFACE......Page 19
ACKNOWLEDGMENTS......Page 23
ABOUT THE AUTHOR......Page 25
1.1 OVER 200 FORMS OF PTM OF PROTEINS......Page 27
1.3.1 Definition and Description of MS......Page 28
1.3.2 Basic Design of Mass Analyzer Instrumentation......Page 29
1.3.3 ESI......Page 33
1.3.4 Nano-ESI......Page 37
1.4 OVERVIEW OF NUCLEIC ACIDS......Page 41
1.5.1 Introduction to Proteomics......Page 46
1.5.2 Protein Structure and Chemistry......Page 48
1.5.3.1 History and Strategy......Page 53
1.5.3.2 Protein Identification through Product Ion Spectra......Page 56
1.5.3.3 High-Energy Product Ions......Page 62
1.5.3.4 De Novo Sequencing......Page 63
1.5.3.5 Electron Capture Dissociation (ECD)......Page 66
1.5.4.2 GP Basicity and Protein Charging......Page 68
1.5.4.3 Calculation of Charge State and Molecular Weight......Page 70
1.5.4.4 Top-Down Protein Sequencing......Page 72
1.5.5 Systems Biology and Bioinformatics......Page 74
1.5.6 Biomarkers in Cancer......Page 78
REFERENCES......Page 82
2.2 BIOLOGICAL PROCESSES OF PROTEIN GLYCOSYLATION......Page 85
2.4 CARBOHYDRATES......Page 86
2.4.1 Ionization of Oligosaccharides......Page 90
2.4.2 Carbohydrate Fragmentation......Page 91
2.4.3 Complex Oligosaccharide Structural Elucidation......Page 96
2.6 GLYCOSYLATION STUDY APPROACHES......Page 98
2.6.1 MS of Glycopeptides......Page 99
2.6.2.1 High Galactose Glycosylation Pattern......Page 101
2.6.5 High-Resolution/High-Mass Accuracy Measurement and Identification......Page 102
2.6.6 Digested Bovine Fetuin......Page 104
REFERENCES......Page 105
3.2 CELLULAR PROCESSES INVOLVED IN SULFATION......Page 107
3.5 FRAGMENTATION NOMENCLATURE FOR CARBOHYDRATES......Page 108
3.6 SULFATED MUCIN OLIGOSACCHARIDES......Page 109
3.7 TYROSINE SULFATION......Page 110
3.8 TYROSYLPROTEIN SULFOTRANSFERASES TPST1 AND TPST2......Page 113
3.10 SULFATED PEPTIDE PRODUCT ION SPECTRA......Page 115
3.11 USE OF HIGHER ENERGY COLLISIONS......Page 119
3.12 ELECTRON CAPTURE DISSOCIATION (ECD)......Page 120
3.13 SULFATION VERSUS PHOSPHORYLATION......Page 121
REFERENCES......Page 123
4.1.2 Protein Phosphatase and Kinase......Page 125
4.1.3 Hydroxy-Amino Acid Phosphorylation......Page 126
4.1.4 Traditional Phosphoproteomic Approaches......Page 128
4.1.5.1 Phosphoproteomic Enrichment Techniques......Page 129
4.1.5.2 IMAC......Page 130
4.1.5.3 MOAC......Page 131
4.1.6 The Ideal Approach......Page 133
4.1.8 Tandem MS Approach......Page 134
4.1.8.1 pS Loss of Phosphate Group......Page 135
4.1.8.2 pT Loss of Phosphate Group......Page 138
4.1.8.3 pY Loss of Phosphate Group......Page 139
4.1.10 Electron Transfer Dissociation (ETD)......Page 141
4.2.2 Background of Study......Page 144
4.2.4.1 Cell Culture......Page 145
4.2.4.4 Solid-Phase Extraction (SPE) Desalting......Page 146
4.2.4.5 Converting Peptide Carboxyl Moieties to Methyl Esters......Page 147
4.2.4.8 In-Gel Reduction, Alkylation, Digestion, and Extraction of Peptides......Page 148
4.2.5.1 RP/Nano-HPLC Separation......Page 149
4.2.6 Current Approaches for Peptide Identification and False Discovery Rate (FDR) Determination......Page 151
4.2.7.1 Detergent Lysis, Trizol, and Ultracentrifugation......Page 152
4.2.7.2 Nucleic Acid Removal with SDS-PAGE......Page 153
4.2.8 HeLa Cell Phosphoproteome Methodology Comparison......Page 154
4.2.8.2 In-Solution and In-Gel Digests Phosphoproteome Coverage......Page 155
4.2.9 Overall Conclusion......Page 160
4.3.1 IMAC Flow Through Peptide Analysis......Page 161
4.3.2 IMAC NaCl Wash Peptide Analysis......Page 162
4.3.4 Gene Ontology Comparison......Page 164
4.3.5 IMAC Bed Nonspecific Binding Study......Page 166
4.4 REVIEWING SPECTRA USING THE SPECTRUMLOOK SOFTWARE PACKAGE......Page 169
REFERENCES......Page 170
5.1.1 Introduction......Page 173
5.1.3.1 PP5......Page 175
5.1.4.1 Producing Cell Cultures......Page 177
5.1.4.2 Protein Extraction......Page 168
5.1.4.3 Phosphopeptide Enrichment by IMAC......Page 180
5.1.4.4 Reversed-Phase (RP)/Nano-HPLC Separation......Page 181
5.1.4.6 Protein Identification and False Discovery Rate (FDR) Determination......Page 182
5.1.4.8 Data Set Peak Matching and Alignment......Page 183
5.1.5 Phosphoproteome Gene Ontology (GO) Comparison......Page 186
5.1.6.1 Analysis of Variance (ANOVA)......Page 188
5.1.6.2 Four Potential Target Proteins......Page 192
5.1.7 GO Differential Comparison......Page 193
5.1.7.3 Molecular Function Interacting Modules......Page 194
5.1.9 Reviewing Spectra Using the SpectrumLook Software Package......Page 201
REFERENCES......Page 202
6.1.3 Caulobacter crescentus......Page 207
6.1.4 Ser/Thr/Tyr Phosphorylation of C. crescentus......Page 209
6.1.5 Ser/Thr/Tyr Phosphorylation of Bacillus subtilis and Escherichia coli......Page 210
6.1.6 C. crescentus as Cell Cycle Model......Page 211
6.1.7 Bacteria Starvation Response......Page 213
6.2.1 Bacterial Strain and Growth Conditions......Page 214
6.2.2 C. crescentus Cell Protein Extraction: Phosphoproteomics......Page 215
6.2.4 In Vitro Methylation of Peptides......Page 216
6.2.5 Phosphopeptide Enrichment by IMAC......Page 217
6.2.8 RP/Nano-High-Performance Liquid Chromatography(HPLC) Separation......Page 218
6.2.11 Peptide Identification and False Discovery Rate (FDR) Determination......Page 219
6.3.1 Total Phosphoprotein Identifications......Page 220
6.3.3 Phosphorylation Sites Identified......Page 222
6.3.4 Ser/Thr/Tyr Phosphoproteome of C. crescentus......Page 231
6.3.5 Phosphorylated His and Aspartate......Page 239
6.3.6 Cell Cycle His Kinase CckA......Page 241
6.3.8.2 TonB-Dependent Receptor Proteins......Page 242
6.3.9.4 Aspartate Phosphorylated Tyr Kinase DivL......Page 243
6.3.10.3 Function of Phosphoproteome of C. crescentus......Page 251
6.3.11.2 Carbon-Starved Environment Phosphorylated Proteins......Page 253
6.3.12.1 Carbon-Rich Mitochondrial Localization......Page 258
6.3.13 Phosphopeptide Quantitative Differential Comparison......Page 259
6.3.13.3 Upregulation NAD-Dependent GDH......Page 260
6.3.14.1 Entire Proteome Localization and Function......Page 261
6.3.14.3 Localization of Regulated Proteins......Page 263
6.3.14.4 Function of Regulated Proteins......Page 264
6.3.14.6 Overlap of Phosphorylated Proteins and Regulated Normal Proteome......Page 265
6.3.14.9 Direct Relationships Observed......Page 266
6.3.16.1 Reviewing Spectra Using the SpectrumLook Software Package......Page 269
REFERENCES......Page 270
7.1 PHOSPHOHISTIDINE AS POSTTRANSLATIONAL MODIFICATION (PTM)......Page 275
7.2 BACTERIAL KINASES AND THE TWO-COMPONENT SYSTEM......Page 276
7.3.1 Stabilities of Phosphorylated Amino Acids......Page 277
7.3.2 Immobilized Metal Affinity Chromatography (IMAC) and Mass Spectrometry (MS)......Page 278
7.4.1 Introduction......Page 281
7.4.2.1 Bacteria Models of Ser/Thr/Tyr Phosphorylation......Page 283
7.4.2.4 Mass Spectral Measurement of Phosphohistidine......Page 284
7.4.3.1 In Vitro Selective pHis Phosphorylation......Page 285
7.4.3.2 In Vitro Phosphorylation of Angio II (Sar1Thr8)......Page 287
7.4.3.4 C. crescentus Cell Protein Extraction with V-8 Protease Digestion......Page 288
7.4.3.5 1-D SDS-Polyacrylamide Gel Electrophoresis (PAGE)......Page 289
7.4.3.6 Phosphohistidine Enrichment by Cu(II)-Based IMAC......Page 290
7.4.3.7 Reversed-Phase (RP)/Nano-HPLC Separation......Page 291
7.4.3.8 Nano-ESI Nano-HPLC MS......Page 292
7.4.4 C18 RP LC Behavior......Page 294
7.4.5 Phosphohistidine Loses HPO3 and H3PO4......Page 296
7.4.5.1 Rational for H3PO4 Loss......Page 298
7.4.6.1 pH-Containing Peptide INpHDLR......Page 303
7.4.6.3 pH-Containing Peptide pHLGLAR......Page 305
7.4.6.4 Singly Charged (1+) Peptide pHLGLAR......Page 306
7.4.7.1 Peptide Angio I as DRVYIHPFHL......Page 307
7.4.8.2 Phosphorylated Angio II as DRVpYIpHPF......Page 311
7.4.9.1 Peptide Angio II (Sar1Thr8)......Page 313
7.4.10 Validation of Cu(II)-Based IMAC Phosphohistidine Enrichment......Page 317
7.4.10.2 Cu(II)-Based IMAC of Angio I......Page 318
7.4.11.1 Time-Based Digestion Study......Page 319
7.4.11.3 Phosphohistidine Product Ion Spectra......Page 320
7.4.12.1 Localization of Phosphorylated Proteins......Page 322
7.4.12.2 Function of Phosphorylated Proteins......Page 330
7.4.13 Predicted Regulatory Protein Motif Study......Page 333
7.4.14.1 Phosphorylation Motif Study......Page 334
7.4.14.2 Phosphohistidine Kinase Motif......Page 335
7.4.15 The pDpH Motif......Page 336
7.5.1 Reviewing Spectra Using the SpectrumLook Software Package......Page 337
REFERENCES......Page 339
APPENDIX I: Atomic Weights and Isotopic Compositions......Page 343
APPENDIX II: Periodic Table of the Elements......Page 351
APPENDIX III: Fundamental Physical Constants......Page 353
GLOSSARY......Page 355
INDEX......Page 371