Ms 339

11 Quantification of Protein-Ligand Interactions in Solution by Hydrogen/ Deuterium Exchange (PLIMSTEX) 342

Mei M. Zhu, David Hambly, and Michael L. Gross

11.1 Introduction 342

11.2 The PLIMSTEX Method 342

11.2.1 A General Protocol of H/D Exchange and LC/MS Analysis for PLIMSTEX 342

11.2.2 Determination and Interpretation of the Titration Curves 343

11.3 Applications of PLIMSTEX 345

11.3.1 Determination of Association Constant (Ka), Stoichiometry (n), and Protection (AD;) 345

11.3.2 Ras-GDP Interacting with Mg2+: A 1:1 Protein:Metal Ion Interaction 347

11.3.2.1 Kinetic Study of Forward H/D Exchange Ras-GDP with Different [Mg2+] 347

11.3.2.2 PLIMSTEX Results for Ras-GDP Titrated with Mg2+ 348

11.3.2.3 Interpretation of PLIMSTEX Results with H/D Exchange Kinetics 349

11.3.2.4 Application of PLIMSTEX to Relatively Weak Protein-Ligand Binding 350

11.3.2.5 Experimental Issues Regarding Using Metal Chelators 350

11.3.3 Apo-CaM Interacting with Ca2+: A 1:4 Protein:Metal Ion Interaction 351

11.3.3.1 PLIMSTEX Results for CaM and Intermediate Protein-Ligand Binding Species 351

11.3.3.2 PLIMSTEX in Biologically Relevant Media and High Ionic Strength 352

11.3.4 Apo-IFABP and Oleate: A Protein-Small Organic Molecule Interaction 353

11.3.5 Holo-CaM and Melittin: A Protein-Peptide Interaction 354 11.3.5.1 PLIMSTEX Curves Under Different Holo-CaM Concentrations 355

11.3.6 Self-association of Insulin: A Protein-Protein Interaction 356 11.3.6.1 Modified Version of PLIMSTEX for Insulin Self-association 356

11.4 Features of PLIMSTEX 357

11.4.1 Determines Stoichiometry, and Protection (AD;) 357

11.4.2 Requires Low Quantities of Protein 357

11.4.3 Relies Only on MS to Measure m/z And Not Solution Concentration 358

11.4.4 Works in Biologically Relevant Media at High Ionic Strength 359

11.4.5 Does Not Need Specially Labeled Protein or Ligand 359

11.4.6 Avoids Perturbation of the Binding Equilibrium 360

11.4.7 Has Potential for Peptide Resolution 360

11.4.8 Current Challenges and Future Directions 360

11.5 Fast Radical Footprinting for Protein-Ligand Interaction Analysis 361

11.5.1 Rationale for Hydroxyl Radicals as a Probe 362

11.5.2 Methods for Generating Hydroxyl Radicals 362

11.5.3 Fast Photochemical Oxidation of Proteins 363

11.5.4 Locating the Sites of Oxidation 364

11.5.5 Application of FPOP to Apomyoglobin 364

11.5.6 Advantages of FPOP 366

11.6 Potential Applications in Drug Discovery 367 References 368

12 Protein-targeting Drug Discovery Guided by Hydrogen/Deuterium Exchange

Mass Spectrometry (DXMS) 377

Yoshitomo Hamuro, Stephen J. Coales, and Virgil L. Woods Jr

12.1 Introduction 377

12.2 Theory of H/D Exchange 378

12.2.1 Amide H/D Exchange 378

12.2.2 Protection Factor 378

12.2.3 Backbone Amide Hydrogens as Thermodynamic Sensors 379

12.3 Overview of H/D Exchange Technologies 380

12.3.1 On Exchange Reaction 380

12.3.2 Quench of Exchange Reaction 380

12.3.3 Protein Fragmentation by Proteolysis 381

12.3.4 Digestion Optimization 381

12.3.5 HPLC Separation 381

12.3.6 Mass Analysis 381

12.3.7 Automation of H/D Exchange by MS 382

12.3.8 Automated Data Analysis 383

12.4 DXMS-guided Design of Well Crystallizing Proteins 383

12.4.1 Disordered Regions and Protein Crystallography 383

12.4.2 Poorly Crystallizing Proteins Contain Substantial Disordered Regions 384

12.4.3 Disorder-depleted Mutant Preserved Ordered Structure 384

12.4.4 Disorder-depleted Mutant Improved Crystallization Efficiency and Produced High Resolution Structure 384

12.5 Rapid Characterization of Protein Conformational Change with DXMS 385

12.5.1 Human Growth Hormone 386

12.5.2 H/D Exchange of hGH 386

12.5.3 Free Energy Change upon Folding of hGH 386

12.6 Application of H/D Exchange to Protein-Small Molecule Ligand Interactions 388

12.6.1 p38 Mitogen-activated Protein Kinase 388

12.6.2 H/D Exchange of p38 MAP Kinase 389

12.6.3 Peroxisome Proliferator-activated Receptor g 390

12.6.4 H/D Exchange of PPARg 390

12.7 DXMS-guided Design of Small Molecules that Target Protein-Protein Interaction Surfaces 391

12.8 Optimal Formulation and Quality Control of Whole-protein Therapeutics with DXMS 393

12.9 Conclusions 394 References 394

V MS in early pharmacokinetics 399

13 Mass Spectrometry in Early Pharmacokinetic Investigations 401

Walter A. Korfmacher

13.1 Introduction 401

13.2 HPLC-MS/MS Overview 402

13.3 In Vitro Applications 405

13.4 In Vivo Applications 406

13.5 Rapid Method Development 408

13.6 Increasing Throughput in HPLC-MS/MS 410

13.7 Matrix Effects 411

13.8 Discovery PK Assay Rules 413

13.9 New Technology in LC-MS 415

13.10 Conclusion 419 References 419

Index 429

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