Hydrogen Exchange Mass Spectrometry of Proteins (eBook, ePUB)
Fundamentals, Methods, and Applications
Redaktion: Weis, David D.
Hydrogen Exchange Mass Spectrometry of Proteins (eBook, ePUB)
Fundamentals, Methods, and Applications
Redaktion: Weis, David D.
- Format: ePub
- Merkliste
- Auf die Merkliste
- Bewerten Bewerten
- Teilen
- Produkt teilen
- Produkterinnerung
- Produkterinnerung
Hier können Sie sich einloggen
Bitte loggen Sie sich zunächst in Ihr Kundenkonto ein oder registrieren Sie sich bei bücher.de, um das eBook-Abo tolino select nutzen zu können.
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,…mehr
- Geräte: eReader
- mit Kopierschutz
- eBook Hilfe
- Größe: 31.42MB
Dieser Download kann aus rechtlichen Gründen nur mit Rechnungsadresse in A, B, BG, CY, CZ, D, DK, EW, E, FIN, F, GR, HR, H, IRL, I, LT, L, LR, M, NL, PL, P, R, S, SLO, SK ausgeliefert werden.
- Produktdetails
- Verlag: John Wiley & Sons
- Seitenzahl: 376
- Erscheinungstermin: 11. Januar 2016
- Englisch
- ISBN-13: 9781118703694
- Artikelnr.: 44503801
- Verlag: John Wiley & Sons
- Seitenzahl: 376
- Erscheinungstermin: 11. Januar 2016
- Englisch
- ISBN-13: 9781118703694
- Artikelnr.: 44503801
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
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