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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.
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.
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.
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.
Über den Autor
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.
Inhaltsverzeichnis
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...
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...
Details
| Erscheinungsjahr: | 2016 |
|---|---|
| Genre: | Chemie |
| Rubrik: | Naturwissenschaften & Technik |
| Medium: | Buch |
| Seiten: | 376 |
| Inhalt: | 376 S. |
| ISBN-13: | 9781118616499 |
| ISBN-10: | 1118616499 |
| Sprache: | Englisch |
| Einband: | Gebunden |
| Autor: | Weis, David D. |
| Redaktion: | Weis, David D |
| Herausgeber: | David D Weis |
| Hersteller: |
John Wiley & Sons
Turner Publishing Company |
| Maße: | 250 x 175 x 27 mm |
| Von/Mit: | David D Weis |
| Erscheinungsdatum: | 21.03.2016 |
| Gewicht: | 0,909 kg |
Über den Autor
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.
Inhaltsverzeichnis
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...
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...
Details
| Erscheinungsjahr: | 2016 |
|---|---|
| Genre: | Chemie |
| Rubrik: | Naturwissenschaften & Technik |
| Medium: | Buch |
| Seiten: | 376 |
| Inhalt: | 376 S. |
| ISBN-13: | 9781118616499 |
| ISBN-10: | 1118616499 |
| Sprache: | Englisch |
| Einband: | Gebunden |
| Autor: | Weis, David D. |
| Redaktion: | Weis, David D |
| Herausgeber: | David D Weis |
| Hersteller: |
John Wiley & Sons
Turner Publishing Company |
| Maße: | 250 x 175 x 27 mm |
| Von/Mit: | David D Weis |
| Erscheinungsdatum: | 21.03.2016 |
| Gewicht: | 0,909 kg |
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