List of Contributors xiii
Foreword by John R. Engen xvii
Preface xix
A Note about Nomenclature xxv
1 Hydrogen Exchange: A Sensitive Analytical Window into Protein Conformation and Dynamics 1
Pernille Foged Jensen and Kasper D. Rand
1.1 Isotopic Exchange and the Study of Protein Conformation and Dynamics 1
1.2 Amide HX in Unstructured Polypeptides 3
1.2.1 Mechanisms of Base- and Acid-Catalyzed Amide HX 4
1.2.2 The Effect of pH and Temperature on Amide HX 6
1.2.3 The Effect of Sequence and Ionic Strength on Amide HX 8
1.2.4 The Effect of Solvent and Pressure on Amide HX 8
1.3 Amide HX in Folded Polypeptides 9
1.3.1 Detecting EX1 and EX2 Kinetics during an HX-MS Experiment 13
References 15
2 Hydrogen Exchange Mass Spectrometry Experimental Design 19
Loo Chien Wang, Srinath Krishnamurthy, and Ganesh Srinivasan Anand
2.1 Application of HX-MS for Protein Dynamics 19
2.1.1 Measuring Conformational Dynamics of Proteins by Hydrogen Exchange 19
2.1.2 Mapping Effects of Perturbations on Protein Dynamics 20
2.2 Factors Governing HX 20
2.2.1 pH 20
2.2.2 Temperature 20
2.2.3 Time 21
2.3 HX-MS Workflow 22
2.3.1 Sample Preparation and Sample Volumes 22
2.3.2 Preparation of Buffer Reconstituted in Deuterium Oxide 24
2.3.3 Preparation and Optimization of Reaction Quench Solution 24
2.3.4 Hydrogen Exchange Reactions 25
2.3.5 Proteolytic Digestion 26
2.3.6 Proteolytic Digest Fragment Identification by Tandem (MS/MS) Mass Spectrometry 27
2.3.7 LC Separation 27
2.3.8 Back-Exchange Consideration 27
2.4 Centroids and Data Analysis 29
2.4.1 Calculation of Centroids of Mass Spectrometric Envelopes 29
2.4.2 Displaying HX-MS Results 33
References 33
3 Data Processing in Bottom-Up Hydrogen Exchange Mass Spectrometry 37
Vladimir Sarpe and David C. Schriemer
3.1 Introduction 37
3.2 The Deuterated Isotopic Distribution 38
3.2.1 Calculating the Average Deuteration 39
3.2.2 Distribution Analysis 40
3.3 Essential Elements of an HX-MS Data Processing Workflow 41
3.3.1 File Import and Project Creation 42
3.3.2 Feature Processing 43
3.3.3 Data Validation 43
3.3.4 Statistical Analysis 43
3.3.5 Visualization 44
3.3.6 Integration 46
3.4 Select Software Packages for Automation of Analysis 46
3.4.1 DynamX 46
3.4.2 HDX Workbench 47
3.4.3 Mass Spec Studio 48
3.4.4 Other Packages 49
3.5 Ongoing and Future Challenges 50
References 51
4 Method Validation and Standards in Hydrogen Exchange Mass Spectrometry 55
Jeffrey W. Hudgens, Richard Y.-C. Huang, and Emma D'Ambro
4.1 Introduction 55
4.2 Rationale for a Reference Measurement System for HX-MS 56
4.3 General Metrological Terminology 58
4.4 Method Validation 58
4.4.1 General Conditions 58
4.4.2 Precision 60
4.4.3 Bias 64
4.4.4 Accuracy Improvements 66
4.4.5 HX-MS and HX-NMR Cross Comparisons 67
4.5 Standards: RM 68
4.6 Summary: Maintaining Standards and Monitoring Performance 69
References 70
5 Millisecond Hydrogen Exchange 73
Derek J. Wilson
5.1 Introduction 73
5.2 Instrumentation 74
5.3 Data Analysis 76
5.3.1 Millisecond HX Kinetics 76
5.3.2 Agreement with Crystal Structure 78
5.4 Applications 79
5.4.1 Millisecond Pulse Labeling for Protein Folding 80
5.4.2 Millisecond Pulse Labeling for Studying Allostery 81
5.4.3 Conformational Dynamics in Weakly Structured Regions of Proteins 84
5.4.4 Dynamics in Active Enzymes 85
5.4.5 Residual Structure in Intrinsically Disordered Proteins 87
5.5 Conclusions and Outlook 87
References 88
6 Proteases for Hydrogen Exchange Mass Spectrometry 93
Eric Forest and Martial Rey
6.1 Introduction 93
6.2 The Use of Pepsin in HX-MS 93
6.2.1 Mechanisms of Proteolysis 94
6.2.2 Specificity 94
6.2.3 Tandem MS and Computer Aids for Mapping 94
6.2.4 Reproducibility 95
6.2.5 Immobilization of Proteases 95
6.2.6 Resolution 95
6.3 The Use of Other Commercially Available Proteases 96
6.4 The Use of Other Acidic Proteases After Expression or Extraction 98
References 104
7 Extracting Information from Hydrogen Exchange Mass Spectrometry Data 107
Zhongqi Zhang and Jing Fang
7.1 Introduction 107
7.2 Basic Concepts in HX Data Analysis 108
7.2.1 Deuterium Incorporation 108
7.2.2 Pseudo First-Order Kinetics and HX Rate Constants 109
7.2.3 Chemical Exchange Rate Constants 109
7.2.4 Protection Factors 110
7.3 Algorithms for Extracting Rate Constants and Protection Factors 110
7.3.1 Back-Exchange Correction 110
7.3.2 Extracting Rate Constants by Nonlinear Curve Fitting 111
7.3.3 Extracting Rate Constants by Semilogarithm Plot 111
7.3.4 Extracting Rate Constant Distributions by Numerical Inverse Laplace Transform 112
7.3.5. Extracting Protection Factors by HX Modeling 114
7.4 Protein Dynamics Hidden in the Isotope Distributions 117
7.4.1 Deconvolution of Natural Isotope Distributions 118
7.4.2 Extracting Kinetic and Thermodynamic Properties of Local Unfolding Dynamics 118
7.5 Concluding Remarks and Future Prospects 123
References 123
8 Gas-Phase Fragmentation of Peptides to Increase the Spatial Resolution of the Hydrogen Exchange Mass Spectrometry Experiment 127
Pernille Foged Jensen and Kasper D. Rand
8.1 Why Increase the Spatial Resolution in an HX Experiment Using MS/MS? 127
8.2 H/D Scrambling in Peptides and How to Avoid It During MS/MS 128
8.2.1 Slow Fragmentation MS/MS Techniques 128
8.2.2 Fast Fragmentation MS/MS Techniques 130
8.2.3 Model Systems for Quantitating Gas-Phase H/D Scrambling 133
8.3 Integrating Gas-Phase Fragmentation Into the Classical Bottom-Up HX-MS Workflow 135
8.3.1 Mass Spectrometers Suitable for an HX-MS/MS Workflow 138
8.3.2 Optimizing the HX-MS/MS Experiment 138
8.3.2.1 Ion Transmission Efficiency 138
8.3.2.2 Spectral Overlap 139
8.3.2.3 Peptide Charge State 139
8.3.2.4 Supplemental Activation 139
8.3.2.5 Targeted HX-MS/MS Acquisition 139
8.3.2.6 Peptide Selection 141
8.4 Recent Applications of the Bottom-Up HX-MS/MS Workflow to Pinpoint the HX Properties of Proteins 141
8.5 Future Directions 143
References 143
9 Top-Down Hydrogen Exchange Mass Spectrometry 149
Igor A. Kaltashov, Rinat R. Abzalimov, Guanbo Wang, and Cedric E. Bobst
9.1 The Appeal of the Top-Down Scheme 149
9.2 Top-Down HX-MS of Small Proteins: The Problem of Hydrogen Scrambling 151
9.2.1 Determinants of Hydrogen Scrambling in Top-Down HX-MS Utilizing Collision-Induced Dissociation of Protein Ions 151
9.2.2 Electron-Based Ion Fragmentation Techniques as a Means of Addressing the Scrambling Problem 152
9.2.3 Top-Down HX ECD (and ETD) MS at Near-Residue Resolution 152
9.3 Conformer-Specific Characterization of Nonnative Protein States Using Top-Down HX ECD MS 156
9.3.1 Characterization of Protein Conformation in an Oligomer-Specific Fashion 156
9.3.2 Characterization of Protein Dynamics in a Conformer-Specific Fashion 157
9.4 Convergence of Top-Down and Classical Schemes of HX-MS: Combination of Proteolytic and Gas-Phase Fragmentation without Chromatographic Separation 158
9.5 The Road Ahead: Challenges and Future Directions 160
Acknowledgments 162
References 162
10 Histidine Hydrogen Exchange for Analysis of Protein Folding, Structure, and Function 165
Michael C. Fitzgerald, Lorrain Jin, and Duc T. Tran
10.1 Introduction 165
10.2 Mechanism of Histidine Hydrogen Exchange 166
10.3 Historical Context 167
10.4 pH-Dependent Experiments with Mass Spectrometry 168
10.4.1 Experimental Workflow 168
10.4.2 Applications 170
10.4.2.1 pKa Analyses Using ESI-MS 170
10.4.2.2 Solvent Accessibility 171
10.4.3 Advantages and Disadvantages 174
10.5 Denaturant-Dependent Experiments 175
10.5.1 Experimental Workflow 176
10.5.2 Applications 177
10.5.2.1 Protein Folding 177
10.5.3 Advantages and Disadvantages 181
10.6 Conclusions and Future Directions 182
Acknowledgment 182
References 182
11 Hydrogen Exchange Mass Spectrometry for the Analysis of Ligand Binding and Protein Aggregation 185
Ying Zhang, Don L. Rempel, and Michael L. Gross
11.1 Protein-Ligand Interactions 185
11.2 Protein-Ligand Affinity Measurements 185
11.3 Conventional Methods for Ligand Binding Characterization 186
11.4 Direct Mass Spectrometry Method 187
11.5 Mass Spectrometry and Hydrogen Exchange 187
11.5.1 HX-MS for Binding Regions 188
11.5.2 HX-MS for Binding Affinity 188
11.6 PLIMSTEX 188
11.6.1 Processing PLIMSTEX Data 190
11.6.2 Examples of PLIMSTEX 193
11.6.3 Advantages of PLIMSTEX 193
11.6.4 Disadvantages of PLIMSTEX 194
11.6.5 Dilution PLIMSTEX (dPLIMSTEX) 197
11.7 SUPREX 198
11.7.1 Examples of SUPREX 200
11.7.2 Advantages of SUPREX 200
11.7.3 Disadvantages of SUPREX 200
11.7.4 HX-MS for Binding Order 201
11.8 HX-MS for Protein-Protein Interactions 201
11.8.1 Self-Association Interactions Using Mass Spectrometry, Self-Titration, and Hydrogen Exchange (SIMSTEX) for Protein Association 201
11.8.2 Pulsed HX for Protein Aggregation 203
11.9 Conclusions 204
Acknowledgment 204
References 204
12 Application of Differential Hydrogen Exchange Mass Spectrometry in Small Molecule Drug Discovery 209
Devrishi Goswami, David P. Marciano, Bruce D. Pascal, Michael J. Chalmers, and Patrick R. Griffin
12.1 Introduction 209
12.2 HX-MS in Drug Discovery 210
12.2.1 Identifying Putative Ligand Binding Sites 210
12.2.1.1 Laulimalide Binding to Microtubule 210
12.2.1.2 Activator Binding to AMP-Activated Protein Kinase 210
12.2.1.3 Small Molecule Binding to VopS, an AMPylator 211
12.2.2 HX Aids in Developing Structure-Activity Relationships 212
12.2.2.1 G Protein-Coupled Receptor Activation by Modulators 213
12.2.2.2 NR PPARgamma Activation by Small Molecules 215
12.2.3 Targeting Intrinsically Disordered Proteins to Aid Drug Discovery 215
12.3 HX in Drug Discovery Requires Automation of the HX Platform 216
12.3.1 The Case for an Automated HX-MS Workflow 216
12.3.2 Decoupled and Real-Time Automation of the HX-MS Experiment 216
12.4 The Need for Statistical Analysis of Differential HX Data 218
12.5 Challenges and Future Directions 219
References 221
13 The Role of Hydrogen Exchange Mass Spectrometry in Assessing the Consistency and Comparability of the Higher-Order Structure of Protein Biopharmaceuticals 225
Damian Houde and Steven A. Berkowitz
13.1 Introduction 225
13.2 Biopharmaceutical Comparability 226
13.3 Internal Comparability (Innovator) versus External Comparability (Biosimilar) 227
13.4 General Challenges in Assessing the Comparability of Biopharmaceuticals in Terms of Their Higher-Order Structure 229
13.5 Higher-Order Structure and HX-MS in the Biopharmaceutical Industry 229
13.6 Challenges and Approaches of Handling Local HX-MS Data 232
13.6.1 Relative Fractional Exchange Comparability Plot 235
13.6.2 Difference Plot 237
13.7 When Is a Difference Real? 238
13.7.1 Criteria for Assessing the Presence of a Difference in HX-MS Comparability Experiments 239
13.8 An Example of HX-MS Data Processing and Display 241
13.9 Using HX-MS to Assess Structure-Function Comparability 242
13.10 The Role of HX-MS in Biopharmaceutical Comparability Studies 242
References 244
14 Utility of Hydrogen Exchange Mass Spectrometry in Epitope Mapping 247
Richard Y.-C. Huang, Adrienne A. Tymiak, and Guodong Chen
14.1 Introduction 247
14.1.1 Rationale for Epitope Mapping 248
14.1.2 Methods for Epitope Mapping 248
14.2 HX-MS Methodology in Epitope Mapping 251
14.2.1 HX-MS Experimental Designs 251
14.2.2 HX-MS Data Interpretation 252
14.2.3 Complementary Strategies 253
14.3 Epitope Mapping Case Studies 254
14.3.1 Protein-Protein Interactions 255
14.3.2 Protein-Peptide Interactions 258
14.4 Conclusions 258
References 259
15 Hydrogen Exchange Mass Spectrometry for Proteins Adsorbed to Solid Surfaces, in Frozen Solutions, and in Amorphous Solids 265
Balakrishnan S. Moorthy, Bo Xie, Jainik P. Panchal, and Elizabeth M. Topp
15.1 Introduction 265
15.2 HX-MS for Proteins Adsorbed to Solid Surfaces 266
15.2.1 Protein Structure and Dynamics at the Solid-Liquid Interface 266
15.2.2 Methods to Study Proteins Adsorbed at the Solid-Liquid Interface 266
15.2.3 Amide HX-MS for Surface-Adsorbed Proteins 267
15.3 HX-MS for Proteins in Frozen Solutions 269
15.3.1 Protein Structure and Dynamics in Frozen Solutions 269
15.3.2 Methods to Study Proteins in Frozen Solutions 269
15.3.3 Amide HX-MS of Proteins in Frozen Solutions 270
15.4 HX-MS for Proteins in Lyophilized Solids 270
15.4.1 Lyophilization and Stability of Therapeutic Proteins 270
15.4.2 Methods to Study Proteins in Lyophilized Solids 271
15.4.3 Solid-State Amide HX-MS 271
15.4.4 Data Analysis and Interpretation 272
15.5 Summary 274
References 274
16 Hydrogen Exchange Mass Spectrometry of Membrane Proteins 279
Eric Forest and Martial Rey
16.1 Introduction 279
16.2 Interaction of Peptides and Proteins with Unilamellar Vesicles Mimicking the Cell Membrane 280
16.2.1 Peptide-Vesicle Interactions 280
16.2.2 Myoglobin-Vesicle Interaction 281
16.2.3 Phospholipase-Vesicle Interaction 281
16.2.4 Diphtheria Toxin-Vesicle Interaction 284
16.3 Integral Membrane Proteins 285
16.3.1 Bovine ADP/ATP Mitochondrial Carrier (bANC1p) 287
16.3.2 ß2-Adrenergic G-Protein-Coupled Receptor (ß2AR) 287
16.3.3 Additional Uses of DDM with Membrane Proteins 290
16.4 Proteins Inserted in Lipid Nanodiscs 291
16.5 Membrane Proteins in Organello 291
16.6 Conclusion 292
References 293
17 Analysis of Disordered Proteins by Hydrogen Exchange Mass Spectrometry 295
David D. Weis
17.1 Intrinsically Disordered Proteins 295
17.1.1 Disorder Prediction 296
17.1.2 Coupled Binding and Folding by Disordered Proteins 298
17.2 Methods to Characterize Disordered Proteins 299
17.3 Applying Hydrogen Exchange Mass Spectrometry to Disordered Proteins 299
17.3.1 Kinetics of Hydrogen Exchange in Disordered Proteins 299
17.3.2 Direct Millisecond Hydrogen Exchange 304
17.3.3 Achieving Millisecond Hydrogen Exchange by Decreasing pH 304
17.3.4 Proteolysis and Peptide Mapping of IDPs 305
17.4 Identifying Disordered Regions with Hydrogen Exchange Mass Spectrometry 306
17.4.1 Apolipoprotein A-I 306
17.4.2 Peroxisome Proliferator-Activated Receptor gamma Coactivator-1alpha 307
17.4.3 Methyl CpG-Binding Protein 2 307
17.4.4 Inhibitor of Nuclear Factor kappaB 307
17.4.5 alpha-Synuclein 307
17.5 Mechanism of Activation of Calcineurin by Calmodulin 308
17.6 CREB-Binding Protein and Activator of Thyroid and Retinoic Acid Receptor: Disordered Proteins that Fold upon Binding 309
17.6.1 Kinetic Analysis of Peptide-Averaged Hydrogen Exchange 310
17.6.2 Hydrogen Exchange in Molten Globular CBP 312
17.6.3 Detection of Residual Helicity in ACTR with Millisecond Hydrogen Exchange 312
17.7 Future Perspectives 316
Acknowledgments 316
References 318
18 Hydrogen Exchange Mass Spectrometry as an Emerging Analytical Tool for Stabilization and Formulation Development of Therapeutic Monoclonal Antibodies 323
Ranajoy Majumdar, C. Russell Middaugh, David D. Weis, and David B. Volkin
18.1 Introduction 323
18.2 Application of the HX-MS Method to mAbs 325
18.3 HX-MS Data Analysis 326
18.4 Case Studies of the Application of HX-MS to Formulation Development of mAbs 326
18.4.1 Impact of Chemical Modifications on mAb Local Dynamics 328
18.4.2 Impact of Environmental Stresses on mAb Local Dynamics 329
18.4.3 Impact of Formulation Additives on mAb Local Dynamics, Conformational Stability, and Aggregation 331
18.5 Identification of Aggregation Hotspots in mAbs Using HX-MS 334
18.6 Challenges and Opportunities for the HX-MS Technique in mAb Formulation Development 336
18.6.1 Analytical Technology Challenges 336
18.6.2 mAb Formulation Development Challenges 337
18.7 Conclusions 338
Acknowledgments 339
References 339
Index 343
