Protein and Peptide Mass Spectrometry in Drug Discovery -- CONTENTS -- PREFACE -- CONTRIBUTORS -- PART I: METHODOLOGY -- 1 Ionization Methods in Protein Mass Spectrometry -- 1.1 History of the Development of Protein Mass Spectrometry -- 1.2 Laser-Based Ionization Methods for Proteins -- 1.2.1 Matrix-Assisted Laser Desorption/Ionization (MALDI) -- 1.2.2 Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization (AP-MALDI) -- 1.2.3 Surface-Enhanced Laser Desorption/Ionization (SELDI) -- 1.2.4 Nanostructure-Initiator Mass Spectrometry (NIMS) -- 1.3 Spray-Based Ionization Methods for Proteins -- 1.3.1 Electrospray Ionization (ESI) -- 1.3.2 Sonic Spray Ionization (SSI) -- 1.3.3 Electrosonic Spray Ionization (ESSI) -- 1.4 Ambient Ionization Methods -- 1.4.1 Desorption Electrospray Ionization (DESI) -- 1.4.2 Fused-Droplet Electrospray Ionization (FD-ESI) -- 1.4.3 Electrospray-Assisted Laser Desorption Ionization (ELDI) -- 1.4.4 Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) -- 1.5 Conclusions -- Acknowledgments -- References -- 2 Ion Activation and Mass Analysis in Protein Mass Spectrometry -- 2.1 Introduction -- 2.1.1 Mass Accuracy -- 2.1.2 Mass Resolving Power -- 2.1.3 Mass Range -- 2.1.4 Scan Speed -- 2.1.5 Tandem MS Analysis -- 2.2 Ion Activation and Tandem MS Analysis -- 2.2.1 Introduction: Fragmentation in Protein MS -- 2.2.2 Collisional Activation Methods -- 2.2.3 Photodissociation -- 2.2.4 Electron-Induced Dissociation -- 2.2.5 Other Radical-Induced Fragmentation Methods -- 2.3 Mass Analyzers -- 2.3.1 Time-of-Flight Mass Analyzer -- 2.3.2 Quadrupole Mass Analyzer and Quadrupole Ion Trap -- 2.3.3 Fourier-Transform Ion Cyclotron Resonance Mass Spectrometer -- 2.3.4 Orbitrap -- 2.3.5 Ion-Mobility Instruments -- References -- 3 Target Proteins: Bottom-up and Top-down Proteomics.

3.1 Mass Spectral Approaches to Targeted Protein Identification -- 3.2 Bottom-up Proteomics -- 3.2.1 Peptide Mass Fingerprinting -- 3.2.2 Bottom-up Proteomics Using Tandem MS: GeLC-MS/MS and Shotgun Digests -- 3.2.3 GeLC-MS/MS -- 3.2.4 Shotgun Digest -- 3.3 Top-down Approaches -- 3.4 Next-Generation Approaches -- References -- 4 Quantitative Proteomics by Mass Spectrometry -- 4.1 Introduction -- 4.2 In-Cell Labeling -- 4.2.1 15N Metabolic Labeling -- 4.2.2 Stable Isotope Labeling by Amino Acid (SILAC) -- 4.3 Quantitation via Isotopic Labeling of Proteins -- 4.3.1 2D PAGE-Based Quantitation -- 4.3.2 Proteolytic Labeling Using 18O Water -- 4.3.3 Quantitative Labeling by Chemical Tagging -- 4.4 Quantitation via Isotopic Labeling on Peptides -- 4.4.1 ICAT -- 4.4.2 iTRAQ -- 4.4.3 SoPIL -- 4.4.4 Absolute Quantitation -- 4.5 Label-Free Quantitation -- 4.6 Conclusions -- Acknowledgment -- References -- 5 Comparative Proteomics by Direct Tissue Analysis Using Imaging Mass Spectrometry -- 5.1 Introduction -- 5.2 Conventional Comparative Proteomics -- 5.3 Comparative Proteomics Using Imaging MS -- 5.3.1 Biomarker Discovery: Breast Cancer -- 5.3.2 Biomarker Discovery: Toxicity -- 5.3.3 Correlating Drug and Protein Distributions -- 5.4 Conclusions -- Acknowledgments -- References -- 6 Peptide and Protein Analysis Using Ion Mobility-Mass Spectrometry -- 6.1 Ion Mobility-Mass Spectrometry: Instrumentation and Separation Selectivity -- 6.1.1 Instrumentation -- 6.1.2 Separation Selectivity in Bioanalyses -- 6.2 Characterizing and Interpreting Peptide and Protein Structures -- 6.2.1 The Motion of Ions within Neutral Gases -- 6.2.2 Considerations for Calculating Collision Cross Sections -- 6.2.3 Computational Approaches for Interpretation of Structure -- 6.3 Applications of IM-MS to Peptide and Protein Characterizations.

6.3.1 Fundamental Studies of Peptide and Protein Ion Structures -- 6.3.2 Studies in Structural Biology-Protein Complex Characterization -- 6.4 Future Directions -- 6.4.1 Applications -- 6.4.2 Instrumentation -- Acknowledgments -- References -- 7 Chemical Footprinting for Determining Protein Properties and Interactions -- 7.1 Introduction to Hydrogen-Deuterium Exchange -- 7.1.1 Fundamentals of Hydrogen-Deuterium Amide Exchange in Proteins -- 7.1.2 EX1 and EX2 Rates of HDX -- 7.2 Experimental Procedures -- 7.2.1 Global Hydrogen-Deuterium Exchange -- 7.2.2 HDX at the Peptide Level -- 7.3 Mass Spectrometry-Based HDX in Practice -- 7.3.1 Protein-Ligand Interactions by Automated HDX -- 7.3.2 Solvent Accessibility by HDX and MALDI-TOF Mass Spectrometry -- 7.3.3 High-Throughput Screening of Protein Ligands by SUPREX -- 7.3.4 Functional Labeling and Multiple Proteases -- 7.3.5 PLIMSTEX: Application in Protein-DNA Interactions -- 7.3.6 HDX and Tandem Mass Spectrometry Analysis -- 7.3.7 Optimizing HDX with High Pressure -- 7.4 Protein Footprinting via Free-Radical Oxidation -- 7.4.1 Fenton Chemistry Oxidation -- 7.4.2 Radiolytic Generation of Hydroxyl Radicals -- 7.4.3 Fast Photochemical Oxidation of Proteins (FPOP) -- 7.4.4 SPROX: Stability of Proteins from Rates of Oxidation -- 7.5 Chemical Crosslinking -- 7.5.1 Drawbacks of Crosslinking -- 7.6 Selective and Irreversible Chemical Modification -- 7.6.1 Acetylation of Lysine -- 7.6.2 Thiol Derivatization of Cysteines -- 7.6.3 Footprinting FMO Protein in Photosynthetic Bacteria -- 7.6.4 Potential Pitfalls -- 7.7 Conclusion -- References -- 8 Microwave Technology to Accelerate Protein Analysis -- 8.1 Introduction -- 8.2 Microwave Technology -- 8.2.1 Application of Microwave Iirradiation to Akabori Reaction -- 8.2.2 Protein Characterization by Microwave Irradiation and MS.

8.2.3 Temperature and Microwave Irradiation Effects on the Enzyme in Protein Digestion -- 8.2.4 Use of Microwave Digestion of Proteins from SDS-PAGE Gels -- 8.2.5 Extraction of Intact Proteins from SDS-PAGE Using Microwave Irradiation -- 8.2.6 Application of Microwave-Assisted Proteolysis Using Trypsin-Immobilized Magnetic Silica Microspheres -- 8.2.7 Acid Hydrolysis of Proteins with Microwave Irradiation -- 8.2.8 Do Protein Denature During Microwave Irradiation? -- 8.3 Summary -- Acknowledgments -- References -- 9 Bioinformatics and Database Searching -- 9.1 Overview -- 9.2 Introduction to Tandem Mass Spectrometry -- 9.2.1 Protein Sequencing -- 9.2.2 Peptide Fragmentation -- 9.3 Overview of Peptide Identification with Database Searching -- 9.4 MyriMatch-IDPicker Protein Identification Pipeline -- 9.4.1 Raw Data File Formats -- 9.4.2 Protein Sequence Databases -- 9.4.3 MyriMatch Database Search Engine -- 9.4.4 Peptide Identification Reporting -- 9.4.5 Post-processing of Search Results Using IDpicker -- 9.5 Results of a Shotgun Proteomics Study -- 9.6 Improvements to MyriMatch Database Search Engine -- 9.6.1 Parallel Processing -- 9.6.2 Protein Modification Analysis -- 9.7 Applications of MyriMatch-IDPicker Pipeline -- 9.7.1 Characterizing Protein-Protein Interactions -- 9.7.2 Characterizing Yeast Proteome on Diverse Instrument Platforms -- 9.7.3 Characterizing DNA-Protein Crosslinks -- 9.8 Conclusions -- Acknowledgments -- References -- PART II: Applications -- 10 Mass Spectrometry-Based Screening and Characterization of Protein-Ligand Complexes in Drug Discovery -- 10.1 Introduction -- 10.2 Affinity Selection Mass Spectrometry (AS-MS) -- 10.2.1 Direct Detection of Noncovalent Protein-Ligand Complexes -- 10.2.2 Indirect Detection of Noncovalent Protein-Ligand Complexes -- 10.3 Solution-Based AS-MS as Screening Technologies.

10.3.1 Automated Ligand Identification System (ALIS) -- 10.3.2 SpeedScreen -- 10.3.3 Ultracentrification Coupled to Mass Spectrometry -- 10.3.4 Gel Filtration-MS Platform -- 10.3.5 Frontal Affinity Chromatography-Mass Spectrometry (FAC-MS) -- 10.3.6 Indirect Detection AS-MS -- 10.3.7 Emerging Technology -- 10.4 Gas-Phase Interactions -- 10.4.1 Ion-Mobility Mass Spectrometry (IMS) -- 10.4.2 Hydrogen-Deuterium Exchange (H/DX) (Including SUPREX and PLIMSTEX) -- 10.4.3 Crosslinking (Including Inhibition of Complex Formation) -- 10.5 Enzyme Activity Assays Using MS for Screening or Confirming Drug Candidates -- 10.5.1 MS to Measure Substrate Turnover -- 10.5.2 Multiple Component Measurements -- 10.5.3 Continuous Flow Screening -- 10.5.4 Immobilized Enzyme Reactor (IMER) -- 10.5.5 Application of MALDI to High-Throughput Enzyme Assays -- 10.5.6 Ratiometric Assays Using MALDI -- 10.5.7 Self-assembled Monolayers for MALDI-MS (SAMDI) -- 10.5.8 Desorption/Ionization Process Off of Porous Silicon (DIOS) and Carbon Nanotubes -- 10.5.9 Overcoming Low Serial Throughput by Rapid Chromatography -- 10.5.10 MALDI-Triple Quadrupole Mass Spectrometry (MALDI-3Q) -- 10.6 Conclusions and Future Directions -- References -- 11 Utilization of Mass Spectrometry for the Structural Characterization of Biopharmaceutical Protein Products -- 11.1 Introduction -- 11.2 MS-Based Approach for the Characterization of Recombinant Therapeutic Proteins -- 11.3 Cell Culture Development -- 11.4 Purification Development -- 11.4.1 Identification of a Pyruvic Acid Modification Covalently Linked at the N-Terminus of a Recombinant IgG4 Fc Fusion Protein -- 11.4.2 Identification of Hinge Region Cleavage in an IgG1 Monoclonal Antibody with Two N-Linked Glycosylation Sites -- 11.5 Formulation Development -- 11.6 Analytical Method Development.

11.6.1 Utilization of Partial Reduction and LC-MS to Distinguish an IgG4 Monoclonal Antibody Charge Variants That Co-elute in Cation Exchange HPLC.