Preface xv
1 Protein Analysis by Shotgun Proteomics 1
Yu Gao and John R. Yates III
1.1 Introduction 1
1.1.1 Terminology 1
1.1.2 Power of Shotgun Proteomics 1
1.1.3 Advantage of Shotgun Proteomics 2
1.2 Overview of Shotgun Proteomics 2
1.3 Sample Preparation 4
1.3.1 Protein Separation 4
1.3.1.1 Overview 4
1.3.1.2 2D-Gel Approach 4
1.3.1.3 Separation of Membrane Protein 5
1.3.1.4 Subcellular Fractionation 5
1.3.1.5 Protein Enrichment 6
1.3.1.6 Phosphoprotein 6
1.3.1.7 Glycoprotein 6
1.3.1.8 AP-MS and Interactome 7
1.3.2 Protein Modification 8
1.3.2.1 Overview 8
1.3.2.2 Reduction of Disulfide Bond and Alkylation 8
1.3.2.3 Chemical Crosslinking 8
1.3.2.4 Proximity Labeling 9
1.3.3 Protein Digestion 9
1.4 Peptide Separation and Data Acquisition 11
1.4.1 Peptide Separation 11
1.4.1.1 Reversed Phase (RP) 11
1.4.1.2 HILIC 11
1.4.1.3 MudPIT 11
1.4.1.4 Capillary Electrophoresis 13
1.4.2 Peptide Ionization 13
1.4.3 Mass Analyzer 13
1.4.4 Peptide Fragmentation Method 15
1.4.4.1 CID/HCD 15
1.4.4.2 ETD/ECD 16
1.4.4.3 IRMPD/UVPD 16
1.4.5 Acquisition Mode 17
1.5 Informatics 17
1.5.1 Peptide Identification 18
1.5.1.1 Database Search 18
1.5.1.2 Spectral Library Search 21
1.5.1.3 De novo Sequencing 22
1.5.1.4 Peptide-Centric Analysis 23
1.5.2 Peptide/Protein Quantitation 23
1.5.2.1 Labeled Quantitation 23
1.5.2.2 Label-Free Quantitation 27
1.5.3 Protein Inference 29
References 31
2 Quantitative Proteomics for Analyses of Multiple Samples in Parallel with Chemical Perturbation 39
Amanda Rae Buchberger, Jillian Johnson, and Lingjun Li
2.1 Introduction 39
2.2 Relative and Absolute Label-Free Quantitation Strategies 40
2.3 Stable Isotope-Based Quantitative Proteomics 42
2.3.1 Relative Quantitation 42
2.3.2 Absolute Quantitation 47
2.4 Conclusion 48
2.5 Methodology 50
2.6 Notes 52
Acknowledgments 55
References 56
3 Chemoproteomic Analyses by Activity-Based Protein Profiling 67
Bryan J. Killinger, Kristoffer R. Brandvold, Susan J. Ramos-Hunter, and Aaron T. Wright
3.1 Introduction 67
3.2 How ABPP Works 68
3.3 ABPP Probe Design 71
3.3.1 Mechanism-Based Probes 72
3.3.2 Reactivity-Based Probes 74
3.3.3 Photoaffinity Probes 74
3.4 ABPP and Mass Spectrometry for Chemoproteomics 75
3.4.1 Determining ABP Target Identity 75
3.4.2 Considerations for Analyzing ABP Targets with MS 77
3.4.3 Determining the Site of ABP Labeling 78
3.4.4 Quantification of ABPP Probe Targets 80
3.4.4.1 Label-Free Methods 80
3.4.4.2 Isotopic Methods 81
3.5 ABPP Applications and Recent Advances 83
3.5.1 Using ABPs for Functional Protein Annotation 83
3.5.2 ABPPs Applied to Microbes and Their Communities 84
3.6 ABPP Applied to Drug Discovery 88
3.7 Comparative, Competitive, and Convolution ABPP 90
3.8 Conclusions and The Outlook of ABPP 91
Acknowledgements 91
References 91
4 Activity-Based Probes for Profiling Protein Activities 101
Kasi V. Ruddraraju and Zhong-Yin Zhang
4.1 Introduction 101
4.2 Design of Activity-Based Probes 102
4.2.1 The Reactive Group 102
4.2.2 The Linker 104
4.2.3 The Tag 104
4.3 Analytical Platforms for ABPP 105
4.3.1 Gel-Based Platforms 105
4.3.2 Mass Spectrometry Platforms for ABPP 106
4.3.3 Microarray Platform for ABPP 107
4.3.4 Capillary Electrophoresis Platform for ABPP 107
4.4 Classes of Enzymes Studied by ABPP 108
4.4.1 Serine Hydrolases 108
4.4.2 Cysteine Proteases 109
4.4.3 Metallohydrolases 110
4.4.4 Glycosidases 111
4.4.5 Protein Kinases 114
4.4.6 Protein Phosphatases 116
4.5 Conclusions 119
Acknowledgment 120
References 120
5 Chemical Probes for Proteins and Networks 127
Scott Lovell, Charlotte L. Sutherell, and Edward W. Tate
5.1 Introduction 127
5.1.1 Probe Design and Validation 128
5.1.2 Application to a Proteomics Workflow 129
5.1.3 Quantitative Chemical Proteomics 131
5.2 Application of Metabolic Chemical Probes to Lipidated Protein Networks 132
5.2.1 Chemical Probes for N-Myristoylation 133
5.2.2 Chemical Probes for Hedgehog Proteins 136
5.3 Chemical Probes for Target Identification 137
5.3.1 Identifying New Target Profiles of Sulforaphane in Breast Cancer Cells 138
5.3.2 Target Profiling of Zerumbone Using a Novel Clickable Probe 140
5.4 Protocol 143
5.4.1 Introduction 143
5.4.2 Materials 143
5.4.2.1 Chemical Tools 143
5.4.2.2 Cell Culture 143
5.4.2.3 Cell Lysis, Enrichment and Sample Preparation 144
5.4.2.4 Click Chemistry and Enrichment 144
5.4.2.5 Proteomics Sample Preparation 144
5.4.2.6 Proteomics Analysis 144
5.4.3 Method 144
5.4.3.1 HeLa Cell Culture and Preparation of Spike-in Standard 144
5.4.3.2 Preparation of Cell Lysates for Protein Enrichment 145
5.4.3.3 Pull-Down Experiments and Sample Preparation 145
5.4.3.4 LC-MS/MS Analysis 147
5.4.3.5 Data Analysis 147
5.4.3.6 Identification of N-Terminal Myristoylated Peptides 151
5.5 Notes 152
References 153
6 Probing Biological Activities with Peptide and Peptidomimetic Biosensors 159
Laura J. Marholz, Tzu-Yi Yang, and Laurie L. Parker
6.1 Introduction 159
6.2 Peptide Biosensors for Assignment and Characterization of Enzymatic Reactions and Substrate Specificity 160
6.3 Screening Inhibitors and Detecting Ligand Interactions 165
6.4 Diagnostic and Clinical Applications 168
6.5 Profiling Enzymatic Activity 172
6.6 Protocol 178
Materials 179
Methods 180
6.7 Conclusion 182
References 182
7 Chemoselective Tagging to Promote Natural Product Discovery 187
Emily J. Tollefson and Erin E. Carlson
7.1 Introduction 187
7.2 Nonreversible Mass Spectrometry Tags 189
7.2.1 Azides and Alkynes 189
7.2.2 Thiols 192
7.2.3 Aminooxy 194
7.3 Reversible Enrichment Tags 195
7.3.1 Boronic Acids 195
7.3.2 Hydrazines 196
7.3.3 Silanes 196
7.3.4 Disulfides 197
7.4 Conclusions 198
7.5 Protocol for Enrichment of Carboxylic-Acid-Containing Natural Products 198
7.5.1 Dialkylsiloxane Resin Synthesis 198
7.5.2 Production of S. rochei Extract 200
7.5.3 Chemoselective Capture 200
7.5.4 Release of Carboxylic-Acid-Containing Compounds from Resin 201
References 201
8 Identification and Quantification of Newly Synthesized Proteins Using Mass-Spectrometry Based Chemical Proteomics 207
Suttipong Suttapitugsakul, Haopeng Xiao, and Ronghu Wu
8.1 Introduction 207
8.2 Protein Labeling to Study Newly Synthesized Proteins 209
8.2.1 Radioactive Labeling 209
8.2.2 Protein Labeling with Fluorescent Probes 209
8.2.3 SILAC Labeling 210
8.2.4 Protein Labeling with Noncanonical Amino Acids 210
8.3 Global Identification of Newly Synthesized Proteins by Noncanonical Amino Acids and MS 212
8.4 Comprehensive Quantification of Newly Synthesized Proteins by MS 213
8.5 Materials 217
8.5.1 Cell Culture and AHA Labeling 217
8.5.2 Cell Lysis 218
8.5.3 Enrichment of Newly Synthesized Proteins Using Click Chemistry 218
8.5.4 On-Bead Protein Reduction, Alkylation, and Digestion 218
8.5.5 Peptide Desalting 218
8.5.6 TMT Labeling 219
8.5.7 Peptide Fractionation 219
8.5.8 StageTips 219
8.5.9 LC-MS/MS Analysis 219
8.5.10 Database Searches and Data Filtering 220
8.6 Methods 220
8.6.1 Cell Culture with AHA Labeling 220
8.6.2 Cell Lysis and Protein Extraction 220
8.6.3 Enrichment of Newly Synthesized Proteins 220
8.6.4 On-Bead Reduction, Alkylation, and Digestion 221
8.6.5 Peptide Desalting 221
8.6.6 TMT Labeling 222
8.6.7 Peptide Fractionation 222
8.6.8 StageTip Purification 222
8.6.9 LC-MS/MS Analysis 223
8.6.10 Database Searches, Data Filtering, and Half-Life Calculation of Newly Synthesized Proteins 223
Acknowledgements 224
References 224
9 Tracing Endocytosis by Mass Spectrometry 231
Mayank Srivastava, Ying Zhang, Linna Wang, and W. Andy Tao
9.1 Introduction 231
9.2 Clathrin-Mediated Endocytosis 232
9.2.1 Proteins Involved in the Formation of Clathrin-Coated Vesicles 233
9.2.2 Molecular Mechanism for CCV Formation 234
9.2.3 Vesicle Uncoating and Fusion with Endosomal Compartments 237
9.3 Mass Spectrometry as a Tool to Study Endocytosis 237
9.3.1 Isolation of Clathrin-Coated Vesicles and Analysis Using Mass Spectrometry 238
9.3.2 Chemical Proteomic Approaches for Studying the Endocytosis 240
9.3.2.1 Identification of Receptor by Ligand-based-Receptor Capture (LRC) Technology 240
9.3.2.2 Studying the Entry and Trafficking of Nanoparticles Using Time-Resolved Chemical Proteomic Approach 241
9.4 Protocols for TITAN 243
9.4.1 Materials 243
9.4.2 Dendrimer Functionalization 245
9.4.2.1 Synthesis of Masked Aldehyde Handle 245
9.4.2.2 Functionalization of Dendrimer 245
9.4.3 Internalization of Dendrimer by HeLa and MS Sample Preparation 247
9.4.4 Mass Spectrometry and Data Analysis 249
9.5 Conclusion and Future Directions 250
References 251
10 Functional Identification of Target by Expression Proteomics (FITExP) 257
Massimiliano Gaetani and Roman A. Zubarev
10.1 Introduction 257
10.2 FITExP Protocol 261
10.2.1 Cell Line(s) and Drugs/Compounds Selection 261
10.2.2 Drug Treatments of Cell Cultures 261
10.2.3 Cell Lysis and Protein Extraction 262
10.2.4 Estimation of Protein Concentration and Protein Sample Processing 263
10.2.5 Protein Digestion 263
10.2.6 Peptide TMT (Tandem Mass Tag) Labeling and Desalting 263
10.2.7 High pH Fractionation TMT 264
10.2.8 Mass Spectrometry Analysis 264
10.2.9 Data Analysis 265
References 265
11 Target Discovery Using Thermal Proteome Profiling 267
Sindhuja Sridharan, Ina Guenthner, Isabelle Becher, Mikhail Savitski, and Marcus Bantscheff
11.1 Introduction 267
11.2 Thermodynamics of Ligand Binding as a Measure of Target Engagement 270
11.3 Thermal Proteome Profiling - Proteome-wide Detection of Drug-Target Interactions 273
11.3.1 Overview 273
11.3.2 Distinguishing Direct Drug Targets from Downstream Effectors of Drug Action 273
11.4 Experimental Formats 275
11.4.1 Temperature-Range Experiment (TPP-TR) 275
11.4.2 Compound Concentration-Range Experiment (TPP-CCR) 277
11.4.3 Two-Dimensional TPP (2D-TPP) 278
11.5 Experimental Protocol 278
11.6 Reagents 280
11.6.1 Step 1: Compound Treatment 280
11.6.2 Step 2: Temperature Treatment 281
11.6.3 Step 3: Protein Digestion and Labeling 282
11.6.4 Step 4: Mass Spectrometric Analysis of Samples 283
11.6.5 Step 5: Peptide and Protein Identification and Quantification 283
11.6.6 Step 6: Data Handling and Analysis 284
11.7 Present Challenges with TPP 284
11.8 CETSA to TPP - Where are We Heading? 285
References 287
12 Chemical Strategies to Glycoprotein Analysis 293
Joseph L. Mertz, Christian Toonstra, and Hui Zhang
12.1 Introduction 293
12.2 Sample Preparation Strategies for Glycoproteomics 297
12.2.1 Enzymatic/Chemical Modification for Glycopeptide Enrichment 297
12.2.2 Enrichment of Glycans or Glycopeptides by Physical-Chemical Approaches 300
12.3 MS Analysis 302
12.3.1 Glycoproteomic Analysis by Mass Spectrometry 302
12.3.2 Bioinformatics and Data Analysis 304
12.4 Conclusions 306
References 307
13 Proteomic Analysis of Protein-Lipid Modifications: Significance and Application 317
Kiall F. Suazo, Garrett Schey, Chad Schaber, Audrey R. Odom John, and Mark D. Distefano
13.1 Introduction 317
13.2 Chemical Proteomic Approach to Identify Lipidated Proteins 318
13.2.1 Fatty Acylation 322
13.2.1.1 N-Myristoylation 323
13.2.1.2 S-Palmitoylation 325
13.2.2 Prenylation 328
13.2.3 Modification with Cholesterol and GPI Anchors 330
13.3 Protocol for Proteomic Analysis of Prenylated Proteins 331
13.3.1 Materials 332
13.3.1.1 Reagents 332
13.3.1.2 Equipment 333
13.3.1.3 Reagents and Instrument Setup 333
13.3.2 Procedure 334
13.3.2.1 Labeling with Probe 334
13.3.2.2 Isolating Parasites via Saponin Lysis 335
13.3.2.3 In-gel Fluorescence Analysis 335
13.3.2.4 Biotinylation and Streptavidin Pull-down 336
13.3.2.5 Sample Preparation for LC-MS/MS Analysis 337
13.3.2.6 LC-MS/MS Analysis 337
13.3.2.7 Proteomic Data Analysis Using Spectral Counting 338
13.3.3 Results 338
References 341
14 Site-Specific Characterization of Asp- and Glu-ADP-Ribosylation by Quantitative Mass Spectrometry 349
Shuai Wang, Yajie Zhang, and Yonghao Yu
14.1 Introduction 349
14.2 Materials 353
14.2.1 Cell Culture 353
14.2.2 Generation of Stable Cell Lines Expressing shPARG 353
14.2.3 Sample Preparation for Mass Spectrometry 353
14.2.4 Mass Spectrometry Analysis 354
14.2.5 Equipment 354
14.3 Methods 354
14.3.1 Generation of shPARG-Expressing Cell Line 354
14.3.2 SILAC Cell Culture 355
14.3.3 Cell Lysis 355
14.3.4 Reduction, Alkylation, and Precipitation of Proteins 355
14.3.5 Protein Digestion and Enrichment of the PARylated Peptides 356
14.3.6 Cleanup of the Peptide 357
14.3.7 Mass Spectrometry Analysis and Data Processing 357
14.4 Notes 357
Acknowledgements 358
References 358
15 MS-Based Hydroxyl Radical Footprinting: Methodology and Application of Fast Photochemical Oxidation of Proteins (FPOP) 363
Ben Niu and Michael L. Gross
15.1 Introduction 363
15.1.1 General Approaches for Mapping Protein Conformations 363
15.1.2 MS-Based Approaches 364
15.2 Generation of Hydroxyl Radicals 365
15.2.1 Fenton and Fenton-like Chemistry 365
15.2.2 Electron-Pulse Radiolysis 368
15.2.3 High-Voltage Electrical Discharge 370
15.2.4 Synchrotron X-ray Radiolysis of Water 371
15.2.5 Plasma Formation of OH Radicals 372
15.2.6 Photolysis of Hydrogen Peroxide 374
15.3 Fast Photochemical Oxidation of Proteins (FPOP) 375
15.3.1 FPOP Footprints Faster than Protein Folding/Unfolding 377
15.3.2 FPOP Dosimetry 378
15.3.3 Primary Radical Lifetime and Adjustment of Radical Scavengers 379
15.3.4 Radical Lifetimes Can Be Milliseconds 381
15.3.5 Differential Scavenging and Use of a Reporter Peptide in FPOP 381
15.3.6 New Reactive Reagents for the FPOP Platform 383
15.4 Applications of FPOP 384
15.4.1 FPOP for Protein-Protein Interactions and Epitope Mapping 384
15.4.2 FPOP for Protein Aggregation/Oligomerization 387
15.4.3 FPOP for Protein Dynamics 390
15.4.4 FPOP for Protein Folding 391
15.4.5 FPOP for Characterizing Membrane Proteins 394
15.5 Conclusions 395
References 396
Index 417
