Hydrogen Exchange Mass Spectrometry of Proteins
Fundamentals, Methods, and Applications

Hydrogen exchange mass spectrometry is widely recognized for its ability to probe the structure and dynamics of proteins. The application of this technique is becoming widespread due to its versatility for providing structural information about challenging biological macromolecules such as antibodies, flexible proteins and glycoproteins. Although the technique has been around for 25 years, this is the first definitive book devoted entirely to the topic.

Hydrogen Exchange Mass Spectrometry of Proteins: Fundamentals, Methods and Applications brings into one comprehensive volume the theory, instrumentation and applications of Hydrogen Exchange Mass Spectrometry (HX-MS) - a technique relevant to bioanalytical chemistry, protein science and pharmaceuticals. The book provides a solid foundation in the basics of the technique and data interpretation to inform readers of current research in the method, and provides illustrative examples of its use in bio- and pharmaceutical chemistry and biophysics

In-depth chapters on the fundamental theory of hydrogen exchange, and tutorial chapters on measurement and data analysis provide the essential background for those ready to adopt HX-MS.  Expert users may advance their current understanding through chapters on methods including membrane protein analysis, alternative proteases, millisecond hydrogen exchange, top-down mass spectrometry, histidine exchange and method validation. All readers can explore the diversity of HX-MS applications in areas such as ligand binding, membrane proteins, drug discovery, therapeutic protein formulation, biocomparability, and intrinsically disordered proteins.

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 PPARγ 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 β2-Adrenergic G-Protein-Coupled Receptor (β2AR) 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 γ Coactivator-1α 307

17.4.3 Methyl CpG-Binding Protein 2 307

17.4.4 Inhibitor of Nuclear Factor κB 307

17.4.5 α-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

Professor David Weis is Assistant Professor of Chemistry at The University of Kansas. B.A., 1993, Earlham College; Ph.D., 1998, Indiana University, Bloomington; Research Assistant Professor, 2004-2006, University of New Mexico, Albuquerque. His areas of specialization is protein mass spectrometry, with research interests in bioanalytical and biophysical chemistry, protein conformation and dynamics, protein-protein and protein-nucleic acid interactions, transcription factors, mass spectrometry, H/D exchange, cross-linking, data analysis software.