Title Page -- Table of Contents -- List of Contributors -- Foreword -- Preface -- References -- A Note about Nomenclature -- References -- 1 Hydrogen Exchange -- 1.1 Isotopic Exchange and the Study of Protein Conformation and Dynamics -- 1.2 Amide HX in Unstructured Polypeptides -- 1.3 Amide HX in Folded Polypeptides -- References -- 2 Hydrogen Exchange Mass Spectrometry Experimental Design -- 2.1 Application of HX-MS for Protein Dynamics -- 2.2 Factors Governing HX -- 2.3 HX-MS Workflow -- 2.4 Centroids and Data Analysis -- References -- 3 Data Processing in Bottom-Up Hydrogen Exchange Mass Spectrometry -- 3.1 Introduction -- 3.2 The Deuterated Isotopic Distribution -- 3.3 Essential Elements of an HX-MS Data Processing Workflow -- 3.4 Select Software Packages for Automation of Analysis -- 3.5 Ongoing and Future Challenges -- References -- 4 Method Validation and Standards in Hydrogen Exchange Mass Spectrometry -- 4.1 Introduction -- 4.2 Rationale for a Reference Measurement System for HX-MS -- 4.3 General Metrological Terminology -- 4.4 Method Validation -- 4.5 Standards: RM -- 4.6 Summary: Maintaining Standards and Monitoring Performance -- References -- 5 Millisecond Hydrogen Exchange -- 5.1 Introduction -- 5.2 Instrumentation -- 5.3 Data Analysis -- 5.4 Applications -- 5.5 Conclusions and Outlook -- References -- 6 Proteases for Hydrogen Exchange Mass Spectrometry -- 6.1 Introduction -- 6.2 The Use of Pepsin in HX-MS -- 6.3 The Use of Other Commercially Available Proteases -- 6.4 The Use of Other Acidic Proteases After Expression or Extraction -- References -- 7 Extracting Information from Hydrogen Exchange Mass Spectrometry Data -- 7.1 Introduction -- 7.2 Basic Concepts in HX Data Analysis -- 7.3 Algorithms for Extracting Rate Constants and Protection Factors -- 7.4 Protein Dynamics Hidden in the Isotope Distributions.

7.5 Concluding Remarks and Future Prospects -- References -- 8 Gas-Phase Fragmentation of Peptides to Increase the Spatial Resolution of the Hydrogen Exchange Mass Spectrometry Experiment -- 8.1 Why Increase the Spatial Resolution in an HX Experiment Using MS/MS? -- 8.2 H/D Scrambling in Peptides and How to Avoid It During MS/MS -- 8.3 Integrating Gas-Phase Fragmentation Into the Classical Bottom-Up HX-MS Workflow -- 8.4 Recent Applications of the Bottom-Up HX-MS/MS Workflow to Pinpoint the HX Properties of Proteins -- 8.5 Future Directions -- References -- 9 Top-Down Hydrogen Exchange Mass Spectrometry -- 9.1 The Appeal of the Top-Down Scheme -- 9.2 Top-Down HX-MS of Small Proteins: The Problem of Hydrogen Scrambling -- 9.3 Conformer-Specific Characterization of Nonnative Protein States Using Top-Down HX ECD MS -- 9.4 Convergence of Top-Down and Classical Schemes of HX-MS: Combination of Proteolytic and Gas-Phase Fragmentation without Chromatographic Separation -- 9.5 The Road Ahead: Challenges and Future Directions -- Acknowledgments -- References -- 10 Histidine Hydrogen Exchange for Analysis of Protein Folding, Structure, and Function -- 10.1 Introduction -- 10.2 Mechanism of Histidine Hydrogen Exchange -- 10.3 Historical Context -- 10.4 pH-Dependent Experiments with Mass Spectrometry -- 10.5 Denaturant-Dependent Experiments -- 10.6 Conclusions and Future Directions -- Acknowledgment -- References -- 11 Hydrogen Exchange Mass Spectrometry for the Analysis of Ligand Binding and Protein Aggregation -- 11.1 Protein-Ligand Interactions -- 11.2 Protein-Ligand Affinity Measurements -- 11.3 Conventional Methods for Ligand Binding Characterization -- 11.4 Direct Mass Spectrometry Method -- 11.5 Mass Spectrometry and Hydrogen Exchange -- 11.6 PLIMSTEX -- 11.7 SUPREX -- 11.8 HX-MS for Protein-Protein Interactions -- 11.9 Conclusions -- Acknowledgment.

References -- 12 Application of Differential Hydrogen Exchange Mass Spectrometry in Small Molecule Drug Discovery -- 12.1 Introduction -- 12.2 HX-MS in Drug Discovery -- 12.3 HX in Drug Discovery Requires Automation of the HX Platform -- 12.4 The Need for Statistical Analysis of Differential HX Data -- 12.5 Challenges and Future Directions -- References -- 13 The Role of Hydrogen Exchange Mass Spectrometry in Assessing the Consistency and Comparability of the Higher-Order Structure of Protein Biopharmaceuticals -- 13.1 Introduction -- 13.2 Biopharmaceutical Comparability -- 13.3 Internal Comparability (Innovator) versus External Comparability (Biosimilar) -- 13.4 General Challenges in Assessing the Comparability of Biopharmaceuticals in Terms of Their Higher-Order Structure -- 13.5 Higher-Order Structure and HX-MS in the Biopharmaceutical Industry -- 13.6 Challenges and Approaches of Handling Local HX-MS Data -- 13.7 When Is a Difference Real? -- 13.8 An Example of HX-MS Data Processing and Display -- 13.9 Using HX-MS to Assess Structure-Function Comparability -- 13.10 The Role of HX-MS in Biopharmaceutical Comparability Studies -- References -- 14 Utility of Hydrogen Exchange Mass Spectrometry in Epitope Mapping -- 14.1 Introduction -- 14.2 HX-MS Methodology in Epitope Mapping -- 14.3 Epitope Mapping Case Studies -- 14.4 Conclusions -- References -- 15 Hydrogen Exchange Mass Spectrometry for Proteins Adsorbed to Solid Surfaces, in Frozen Solutions, and in Amorphous Solids -- 15.1 Introduction -- 15.2 HX-MS for Proteins Adsorbed to Solid Surfaces -- 15.3 HX-MS for Proteins in Frozen Solutions -- 15.4 HX-MS for Proteins in Lyophilized Solids -- 15.5 Summary -- References -- 16 Hydrogen Exchange Mass Spectrometry of Membrane Proteins -- 16.1 Introduction -- 16.2 Interaction of Peptides and Proteins with Unilamellar Vesicles Mimicking the Cell Membrane.

16.3 Integral Membrane Proteins -- 16.4 Proteins Inserted in Lipid Nanodiscs -- 16.5 Membrane Proteins in Organello -- 16.6 Conclusion -- References -- 17 Analysis of Disordered Proteins by Hydrogen Exchange Mass Spectrometry -- 17.1 Intrinsically Disordered Proteins -- 17.2 Methods to Characterize Disordered Proteins -- 17.3 Applying Hydrogen Exchange Mass Spectrometry to Disordered Proteins -- 17.4 Identifying Disordered Regions with Hydrogen Exchange Mass Spectrometry -- 17.5 Mechanism of Activation of Calcineurin by Calmodulin -- 17.6 CREB-Binding Protein and Activator of Thyroid and Retinoic Acid Receptor: Disordered Proteins that Fold upon Binding -- 17.7 Future Perspectives -- Acknowledgments -- References -- 18 Hydrogen Exchange Mass Spectrometry as an Emerging Analytical Tool for Stabilization and Formulation Development of Therapeutic Monoclonal Antibodies -- 18.1 Introduction -- 18.2 Application of the HX-MS Method to mAbs -- 18.3 HX-MS Data Analysis -- 18.4 Case Studies of the Application of HX-MS to Formulation Development of mAbs -- 18.5 Identification of Aggregation Hotspots in mAbs Using HX-MS -- 18.6 Challenges and Opportunities for the HX-MS Technique in mAb Formulation Development -- 18.7 Conclusions -- Acknowledgments -- References -- Index -- End User License Agreement.