PREFACE ........................................................ xv
CONTRIBUTORS ................................................. xvii
PART I METHODOLOGY ............................................. 1
1 Ionization Methods in Protein Mass Spectrometry .............. 3
Ismael Cotte-Rodriguez, Yun Zhang, Zhixin Miao, and Hau Chen
1.1 History of the Development of Protein Mass
Spectrometry ............................................ 4
1.2 Laser-Based Ionization Methods for Proteins ............. 5
1.2.1 Matrix-Assisted Laser Desorption/Ionization
(MALDI) ........................................... 5
1.2.2 Atmospheric Pressure Matrix-Assisted Laser
Desorption/Ionization (AP-MALDI) .................. 8
1.2.3 Surface-Enhanced Laser Desorption/Ionization
(SELDI) ........................................... 9
1.2.4 Nanostructure-Initiator Mass Spectrometry
(NTMS) ........................................... 11
1.3 Spray-Based Ionization Methods for Proteins ............ 13
1.3.1 Electrospray Ionization (ESI) .................... 13
1.3.2 Sonic Spray Ionization (SSI) ..................... 14
1.3.3 Electrosonic Spray Ionization (ESSI) ............. 17
1.4 Ambient Ionization Methods ............................. 20
1.4.1 Desorption Electrospray Ionization (DESI) ........ 21
1.4.2 Fused-Droplet Electrospray Ionization (FD-ESI) ... 24
1.4.3 Electrospray-Assisted Laser Desorption
Ionization (ELDI) ................................ 27
1.4.4 Matrix-Assisted Laser Desorption Electrospray
Ionization (MALDESI) ............................. 30
1.5 Conclusions ............................................ 30
Acknowledgments ............................................. 30
References .................................................. 30
2 Ion Activation and Mass Analysis in Protein Mass
Spectrometry ................................................ 43
Cheng Lin and Peter O'Connor
2.1 Introduction ........................................... 43
2.1.1 Mass Accuracy .................................... 43
2.1.2 Mass Resolving Power ............................. 44
2.1.3 Mass Range ....................................... 44
2.1.4 Scan Speed ....................................... 45
2.1.5 Tandem MS Analysis ............................... 46
2.2 Ion Activation and Tandem MS Analysis .................. 46
2.2.1 Introduction: Fragmentation in Protein MS ........ 46
2.2.2 Collisional Activation Methods ................... 48
2.2.3 Photodissociation ................................ 50
2.2.4 Electron-Induced Dissociation .................... 55
2.2.5 Other Radical-Induced Fragmentation Methods ...... 59
2.3 Mass Analyzers ......................................... 59
2.3.1 Time-of-Flight Mass Analyzer ..................... 60
2.3.2 Quadrupole Mass Analyzer and Quadrupole Ion
Trap ............................................. 66
2.3.3 Fourier-Transform Ion Cyclotron Resonance Mass
Spectrometer ..................................... 73
2.3.4 Orbitrap ......................................... 77
2.3.5 Ion-Mobility Instruments ......................... 80
References .................................................. 81
3 Target Proteins: Bottom-up and Top-down Proteomics .......... 89
Michael Buync and Ron Base
3.1 Mass Spectral Approaches to Targeted Protein
Identification ......................................... 89
3.2 Bottom-up Proteomics ................................... 90
3.2.1 Peptide Mass Fingerprinting ...................... 91
3.2.2 Bottom-up Proteomics Using Tandem MS:
GeLC-MS/MS and Shotgun Digests ................... 91
3.2.3 GeLC-MS/MS ....................................... 93
3.2.4 Shotgun Digest ................................... 94
3.3 Top-down Approaches .................................... 96
3.4 Next-Generation Approaches ............................. 98
References .................................................. 99
4 Quantitative Proteomics by Mass Spectrometry ............... 101
Jacob Galan, Anton lliuk, and W. Andy Tao
4.1 Introduction .......................................... 101
4.2 In-Cell Labeling ...................................... 105
4.2.1 15N Metabolic Labeling .......................... 105
4.2.2 Stable Isotope Labeling by Amino Acid (SILAC) ... 106
4.3 Quantitation via Isotopic Labeling of Proteins ........ 107
4.3.1 2D PAGE-Based Quantitation ...................... 108
4.3.2 Proteolytic Labeling Using 18O Water ............ 109
4.3.3 Quantitative Labeling by Chemical Tagging ....... 110
4.4 Quantitation via Isotopic Labeling on Peptides ........ 112
4.4.1 ICAT ............................................ 112
4.4.2 iTRAQ ........................................... 113
4.4.3 SoPIL ........................................... 113
4.4.4 Absolute Quantitation ........................... 114
4.5 Label-Free Quantitation ............................... 116
4.6 Conclusions ........................................... 119
Acknowledgment ............................................. 120
References ................................................. 120
5 Comparative Proteomics by Direct Tissue Analysis Using
Imaging Mass Spectrometry .................................. 129
Michelle L. Reyzer and Richard M. Caprioli
5.1 Introduction .......................................... 129
5.2 Conventional Comparative Proteomics ................... 130
5.3 Comparative Proteomics Using Imaging MS ............... 131
5.3.1 Biomarker Discovery: Breast Cancer .............. 131
5.3.2 Biomarker Discovery: Toxicity ................... 133
5.3.3 Correlating Drug and Protein Distributions ...... 134
5.4 Conclusions ........................................... 136
Acknowledgments ............................................ 137
References ................................................. 137
6 Peptide and Protein Analysis Using Ion Mobility-Mass
Spectrometry ............................................... 139
Jeffrey R. Enders, Michal Kliman,
Sevugarajan Sundarapandian, and John A. McLean
6.1 Ion Mobility-Mass Spectrometry: Instrumentation and
Separation Selectivity ................................ 139
6.1.1 Instrumentation ................................. 140
6.1.2 Separation Selectivity in Bioanalyses ........... 145
6.2 Characterizing and Interpreting Peptide and Protein
Structures ............................................ 147
6.2.1 The Motion of Ions within Neutral Gases ......... 147
6.2.2 Considerations for Calculating Collision Cross
Sections ........................................ 148
6.2.3 Computational Approaches for Interpretation of
Structure ....................................... 149
6.3 Applications of IM-MS to Peptide and Protein
Characterizations ..................................... 152
6.3.1 Fundamental Studies of Peptide and Protein Ion
Structures ...................................... 152
6.3.2 Studies in Structural Biology—Protein Complex
Characterization ................................ 157
6.4 Future Directions ..................................... 158
6.4.1 Applications .................................... 158
6.4.2 Instrumentation ................................. 159
Acknowledgments ............................................ 159
References ................................................. 160
7 Chemical Footprinting for Determining Protein Properties
and Interactions ........................................... 175
Sandra A. Kerfoot and Michael L. Gross
7.1 Introduction to Hydrogen-Deuterium Exchange ........... 175
7.1.1 Fundamentals of Hydrogen-Deuterium Amide
Exchange in Proteins ............................ 176
7.1.2 EX 1 and EX2 Rates of HDX ....................... 176
7.2 Experimental Procedures ............................... 178
7.2.1 Global Hydrogen-Deuterium Exchange .............. 178
7.2.2 HDX at the Peptide Level ........................ 179
7.3 Mass Spectrometry-Based HDX in Practice ............... 182
7.3.1 Protein-Ligand Interactions by Automated HDX .... 182
7.3.2 Solvent Accessibility by HDX and MALDI-TOF
Mass Spectrometry ............................... 183
7.3.3 High-Throughput Screening of Protein Ligands
by SUPREX ....................................... 184
7.3.4 Functional Labeling and Multiple Proteases ...... 188
7.3.5 PLIMSTEX: Application in Protein-DNA
Interactions .................................... 188
7.3.6 HDX and Tandem Mass Spectrometry Analysis ....... 191
7.3.7 Optimizing HDX with High Pressure ............... 192
7.4 Protein Footprinting via Free-Radical Oxidation ....... 193
7.4.1 Fenton Chemistry Oxidation ...................... 194
7.4.2 Radiolytic Generation of Hydroxyl Radicals ...... 196
7.4.3 Fast Photochemical Oxidation of Proteins
(FPOP) .......................................... 197
7.4.4 SPROX: Stability of Proteins from Rates of
Oxidation ....................................... 198
7.5 Chemical Crosslinking ................................. 198
7.5.1 Drawbacks of Crosslinking ....................... 199
7.6 Selective and Irreversible Chemical Modification ...... 201
7.6.1 Acetylation of Lysine ........................... 202
7.6.2 Thiol Derivatization of Cysteines ............... 203
7.6.3 Footprinting FMO Protein in Photosynthetic
Bacteria ........................................ 203
7.6.4 Potential Pitfalls .............................. 205
7.7 Conclusion ............................................ 205
References ................................................. 206
8 Microwave Technology to Accelerate Protein Analysis ........ 213
Urooj A. Mirza, Birendra N. Pramanik, and Ajay K. Base
8.1 Introduction .......................................... 213
8.2 Microwave Technology .................................. 215
8.2.1 Application of Microwave Iirradiation to
Akabori Reaction ................................ 215
8.2.2 Protein Characterization by Microwave
Irradiation and MS .............................. 216
8.2.3 Temperature and Microwave Irradiation Effects
on the Enzyme in Protein Digestion .............. 217
8.2.4 Use of Microwave Digestion of Proteins from
SDS-PAGEGels .................................... 219
8.2.5 Extraction of Intact Proteins from SDS-PAGE
Using Microwave Irradiation ..................... 219
8.2.6 Application of Microwave-Assisted Proteolysis
Using Trypsin-Immobilized Magnetic Silica
Microspheres .................................... 220
8.2.7 Acid Hydrolysis of Proteins with Microwave
Irradiation ..................................... 221
8.2.8 Do Protein Denature During Microwave
Irradiation? .................................... 222
8.3 Summary ............................................... 224
Acknowledgments ............................................ 224
References ................................................. 224
9 Bioinformatics and Database Searching ...................... 231
Surendra Dasari and David L. Tabb
9.1 Overview .............................................. 231
9.2 Introduction to Tandem Mass Spectrometry .............. 231
9.2.1 Protein Sequencing .............................. 231
9.2.2 Peptide Fragmentation ........................... 232
9.3 Overview of Peptide Identification with Database
Searching ............................................. 234
9.4 MyriMatch-IDPicker Protein Identification Pipeline .... 235
9.4.1 Raw Data File Formats ........................... 235
9.4.2 Protein Sequence Databases ...................... 237
9.4.3 MyriMatch Database Search Engine ................ 239
9.4.4 Peptide Identification Reporting ................ 242
9.4.5 Post-processing of Search Results Using
IDpicker ........................................ 243
9.5 Results of a Shotgun Proteomics Study ................. 246
9.6 Improvements to MyriMatch Database Search Engine ...... 248
9.6.1 Parallel Processing ............................. 248
9.6.2 Protein Modification Analysis ................... 249
9.7 Applications of MyriMatch-IDPicker Pipeline ........... 250
9.7.1 Characterizing Protein-Protein Interactions ..... 250
9.7.2 Characterizing Yeast Proteome on Diverse
Instrument Platforms ............................ 250
9.7.3 Characterizing DNA-Protein Crosslinks ........... 250
9.8 Conclusions ........................................... 251
Acknowledgments ............................................ 251
References ................................................. 251
PART II Applications ......................................... 253
10 Mass Spectrometry-Based Screening and Characterization
of Protein-Ligand Complexes in Drug Discovery .............. 255
11 Utilization of Mass Spectrometry for the Structural
Characterization of Biopharmaceutical Protein Products ..... 287
Amareth Lim and Catherine A. Srebalus Barnes
11.1 Introduction .......................................... 287
11.2 MS-Based Approach for the Characterization of
Recombinant Therapeutic Proteins ...................... 288
11.3 Cell Culture Development .............................. 290
11.4 Purification Development .............................. 294
11.4.1 Identification of a Pyruvic Acid Modification
Covalently Linked at the N-Terminus of a
Recombinant IgG4 Fc Fusion Protein ............. 295
11.4.2 Identification of Hinge Region Cleavage in an
IgGl Monoclonal Antibody with Two N-Linked
Glycosylation Sites ............................ 298
11.5 Formulation Development ............................... 300
11.6 Analytical Method Development ......................... 304
11.6.1 Utilization of Partial Reduction and LC-MS to
Distinguish an IgG4 Monoclonal Antibody
Charge Variants That Co-elute in Cation
Exchange HPLC .................................. 304
11.6.2 Development of an RP-HPLC Method for
Monitoring an IgG4 Fc Fusion Protein
Post-Translational Modifications ............... 309
11.7 Confirmation of Structure/Product Comparability
Assessment ............................................ 311
11.8 Conclusions ........................................... 313
Acknowledgments ............................................ 315
References ................................................. 315
12 Post-translationally Modified Proteins: Glycosylation,
Phosphorylation, and Disulfide Bond Formation .............. 321
Anthony Tsarbopoulos and Fotini N. Bazoti
12.1 Introduction .......................................... 321
12.2 Glycosylation ......................................... 322
12.2.1 MS Detection of Glycoproteins .................. 323
12.2.2 Glycan Identification, Classification, and
Heterogeneity .................................. 327
12.2.3 Glycoprotein Mapping by LC-ESI and MALDI
Tandem MS ...................................... 329
12.2.4 Glycosylation Site Quantitation ................ 336
12.3 Phosphorylation ....................................... 338
12.3.1 MS Detection of Phosphorylation ................ 338
12.3.2 Enrichment of Phosphorylated Peptides and
Proteins ....................................... 340
12.3.3 Phosphorylation Site Identification ............ 341
12.3.4 Phosphopeptide Quantitation .................... 346
12.4 Disulfide Bond Detection and Mapping .................. 347
12.4.1 MS Detection ................................... 347
12.4.2 Disulfide Mapping .............................. 347
12.5 Future Perspectives ................................... 350
Acknowledgments ............................................ 352
Abbreviations .............................................. 353
References ................................................. 354
13 Mass Spectrometry of Antigenic Peptides .................... 371
Henry Rohrs
13.1 Introduction .......................................... 371
13.1.1 Brief History of MHC Studies ................... 371
13.1.2 Brief Introduction to Immunobiology ............ 372
13.2 Analysis of Antigenic Peptides ........................ 374
13.2.1 MHC Peptide Analysis in Practice—Sample
Preparation .................................... 376
13.2.2 MHC Peptide Analysis in Practice—HPLC
Separation ..................................... 377
13.2.3 MHC Peptide Analysis in Practice—Mass
Spectrometers .................................. 377
13.2.4 MHC Peptide Analysis in Practice—Data
Analysis ....................................... 379
13.3 Examples of the Application of Mass Spectrometry to
Antigenic Peptide Study ............................... 381
13.3.1 Work of D. Hunt ................................ 381
13.3.2 Work of E. Unanue .............................. 382
13.3.3 Work of H. Rammensee ........................... 384
13.3.4 Work of P. Allen ............................... 384
13.3.5 Work of P. Thibault ............................ 385
13.4 Future Work ........................................... 385
Acknowledgments ............................................ 386
Abbreviations .............................................. 387
References ................................................. 387
14 Neuropeptidomics ........................................... 393
Jonathan V. Sweedler, Fang Xie, and Adriana Bora
14.1 Introduction .......................................... 393
14.2 Neuropeptidomics: Characterizing Peptides in the
Brain ................................................. 394
14.3 Sample Preparation for Mass Spectrometry .............. 395
14.3.1 Direct Tissue Profiling ........................ 397
14.3.2 Extraction-Based Strategies .................... 399
14.3.3 Collecting Peptide Release ..................... 400
14.3.4 Sample Preparation for MSI ..................... 403
14.4 Separations ........................................... 405
14.5 Peptide Characterization via Mass Spectrometry ........ 407
14.5.1 Qualitative Analyses ........................... 407
14.5.2 Relative Quantitative Analyses ................. 413
14.5.3 Data Analysis with Bioinformatics .............. 416
14.6 Conclusions ........................................... 419
14.7 Future Perspectives ................................... 419
Acknowledgments ............................................ 420
References ................................................. 420
15 Mass Spectrometry for the Study of Peptide Drug
Metabolism ................................................. 435
Patrick J. Rudewicz
15.1 Introduction .......................................... 435
15.2 Peptide Drug Metabolism ............................... 436
15.3 LC-MS/MS for Metabolite Identification ................ 437
15.4 Quantitative Analysis ................................. 439
15.5 Case Study: 1L-1/β Protease Inhibitors ................ 440
15.6 Future Directions ..................................... 445
References ................................................. 445
INDEX ......................................................... 449
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