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  Contents  
 
  Preface XV
  A Personal Foreword XVII
  List of Contributors XIX
I Introduction to MS in bioanalysis 1
1 Mass Spectrometry in Bioanalysis -- Methods, Principles and Instrumentation
Gérard Hopfgartner 		3
1.1 Introduction 3
1.2 Fundamentals 4
1.3 Ionization Techniques 10
1.3.1 Electron Impact and Chemical Ionization 10
1.3.2 Atmospheric Pressure Ionization 12
1.3.2.1 Electrospray 14
1.3.2.2 Atmospheric Pressure Chemical Ionization 17
1.3.2.3 Photoionization 19
1.3.2.4 Multiple Ionization Source 19
1.3.2.5 Desorption Electrospray and Direct Analysis in Real Time 20
1.3.3 Matrix Assisted Laser Desorption Ionization 21
1.4 Mass Analyzers 23
1.4.1 Quadrupole Analyzers 23
1.4.2 Triple Quadrupole Mass Analyzer 24
1.4.3 Ion Trap Mass Spectrometry 27
1.4.4 Triple Quadrupole Linear Ion Trap 30
1.4.5 Time of Flight Mass Spectrometry 33
1.4.6 Fourier Transform Mass Spectrometry 36
1.4.6.1 Fourier Transform--Ion Cyclotron Resonance Mass Spectrometry 36
1.4.6.2 Orbitrap Mass Spectrometer 37
1.5 Ion Detectors 38
1.6 Practical Aspects and Applications in Bioanalysis 41
1.6.1 Introduction 41
1.6.2 Quantitative Analysis in Biological Matrices 42
1.6.3 Drug Metabolism 45
1.6.4 Analysis of Proteins 49
1.7 Perspectives 54
1.8 Common Definitions and Abbreviations 58
References 58
II Studying target-ligand interactions analyzing the ligand by MS 63
2 Drug Screening Using Gel Permeation Chromatography Spin Columns Coupled with ESI-MS
Marshall M. Siegel 		65
2.1 Introduction 65
2.1.1 Preface 65
2.1.2 Direct and Indirect ESI-MS Analysis of Non-covalent Drug--Protein Complexes 65
2.1.3 Advantages of GPC Spin Columns 66
2.1.4 Application of Equilibrium and Non-equilibrium Theory for the Analysis of GPC Spin Column Eluates 68
2.1.4.1 Sample Prepared Under Equilibrium Conditions Prior to Spin Column Treatment 69
2.1.4.2 Calculation for Predicting the Concentration of Sample Complex Eluted From the Spin Column 69
2.1.4.3 Estimation of Relative Binding Affinities from GPC Spin-Column/ESI-MS Data 72
2.1.4.4 Experimental Determination of the Kd Value from GPC Spin-Column/ESI-MS Data 72
2.2 Experimental 73
2.2.1 Spin Columns 73
2.2.2 Spin Column Media: Advantages and Disadvantages, Volatile vs Non-volatile Buffers 74
2.2.3 Preparing Non-covalent Complexes in Protein Buffer; Protein Concentration, Ligand Concentration, Incubation Time 75
2.2.4 Sample Organization: Single Samples vs Mixtures, Mixture Set-up: Compatibility of Components, Plate Set-up 79
2.2.5 Pooling Spin Column Eluates for Higher Throughput 80
2.2.6 Manual vs Robotic Instrumentation for Sample Preparation and Acquiring Spin Column Eluates 80
2.2.7 ESI Mass Spectrometer: ESI, APCI, Photodissociation, Positive/Negative Ionization 81
2.2.8 ESI Multi-sprayer (MUX) Technology; Sample Throughput; Protein Consumption 82
2.2.9 Reversed Phase (RP) HPLC ESI-MS Considerations 83
2.2.10 Protein Removal for Optimum Sensitivity 84
2.2.11 Data Reduction and Automated Interpretation of GPC Spin Column/ESI-MS Data 84
2.3 Results 89
2.3.1 Secondary Screens 89
2.3.1.1 GPC Spin Column/ESI-MS Drug Screening Demonstration Papers 89
2.3.1.2 Estrogen Receptor Target 89
2.3.1.3 Non-covalent Binding of Drugs to RNA/DNA Targets 90
2.3.1.4 Amgen Secondary Screens 94
2.3.1.5 Novartis Secondary Screens 94
2.3.2 Primary Screens 94
2.3.2.1 RGS4 Protein Target 94
2.3.2.2 Amgen Primary Screens 98
2.3.2.3 Novartis Primary Screens 98
2.3.3 Additional Spin Column Methods 99
2.3.3.1 Competition Experiments of Inhibitor Mixture with Protein Target 99
2.3.3.2 GPC Spin Column/ESI-MS Determination of Binding Sites 101
2.3.3.3 Obtaining MS EC50s and Kds for Ligands Non-covalently Bound to Protein Active Sites 112
2.3.3.4 Multiple Passes Through Spin Columns -- Finding Strongest Binders 113
2.3.3.5 Reverse Screening with GPC Spin Columns 113
2.4 Conclusions 113
2.4.1 GPC Spin Column/ESI-MS: Ease of Use, Mixture Analysis, High Speed, Reliability, Uncoupling of GPC from ESI-MS and HPLC ESI-MS 113
2.4.2 Comparison of GPC Spin Column/HPLC ESI-MS with Tandem Chromatographic Method of GPC/HPLC ESI-MS 114
2.4.3 Future Developments 115
2.4.3.1 MS and HPLC Improvements 115
2.4.3.2 Use of Automated Nanospray for Greater Sensitivity and Smaller Sample Size (Less Protein/Drug) 115
2.4.3.3 Microfluidic Systems: Sensitivity, High Speed 116
2.4.3.4 GPC Spin Column Eluates Analyzed by ESI/Ion Mobility/Mass Spectrometry 116
2.4.3.5 GPC Spin Columns with Matrixless MALDI-MS and Gyros GPC Microfluidic ESI/MALDI-MS System 116
References 117
3 ALIS: An Affinity Selection--Mass Spectrometry System for the Discovery and Characterization of Protein--Ligand Interactions
Allen Annis, Cheng-Chi Chuang, and Naim Nazef 		121
3.1 Introduction 121
3.1.1 State of the Art 122
3.1.1.1 Spectroscopic and Biophysical Methods 122
3.1.1.2 Mass Spectrometry-based Methods 123
3.2 ALIS: An Affinity Selection--Mass Spectrometry System based on Continuous SEC 124
3.2.1 ALIS System Design 126
3.3 Discovery of Ligands from Combinatorial Libraries 127
3.4 Quantitative Binding Affinity Measurement 130
3.4.1 Theory 131
3.4.2 Simulations and Experimental Results 134
3.5 Competition-based Binding Site Determination and Affinity Ranking in Mixtures 135
3.5.1 Binding Site Classification 136
3.5.2 Affinity Ranking in Compound Mixtures 140
3.6 Protein--Ligand Dissociation Rate Measurement 142
3.6.1 Theory 143
3.6.2 Simulations 145
3.6.3 Experimental Results 147
3.7 Conclusions 150
3.8 Future Directions 151
References 152
4 Library Screening Using Ultrafiltration and Mass Spectrometry
Timothy E. Cloutier and Kenneth M. Comess 		157
4.1 Introduction 157
4.2 Ultra-high Throughput Filtration-based Affinity Screening as a Discovery Tool 163
4.2.1 Affinity Selection/Mass Spectrometry 163
4.2.2 Primary Screening Strategy 164
4.2.3 Retesting and Deconvolution Strategy 167
4.2.4 Promiscuous Compound Filter 168
4.2.5 MurF Lead Discovery 171
4.3 Additional Affinity Screening Methodology That Includes Mass Spectrometry-based Readout 177
4.3.1 Pulsed Ultrafiltration MS 177
4.4 Conclusions and Future Directions 180
References 181
5 Continuous-flow Systems for Ligand Binding and Enzyme Inhibition Assays Based on Mass Spectrometry
Hubertus Irth 		185
5.1 Introduction 185
5.2 Continuous-flow Enzyme Assays Based on Mass Spectrometry 186
5.2.1 Assay Principle 186
5.2.2 ESI-MS Assay of Cathepsin B 188
5.2.2.1 MS Assay Development for Cathepsin B 188
5.2.2.2 Compatibility of Cathepsin B Assay with MS Detection 188
5.2.2.3 On-line Coupling of MS-based Cathepsin B Assay to HPLC 190
5.2.2.4 Screening of Natural Products for Cathepsin B Activity 192
5.2.3 ESI-MS Assay of Acetylcholinesterase 194
5.2.3.1 MS Assay Development for Acetylcholinesterase 194
5.2.3.2 Assay Validation and Stability 197
5.2.3.3 Screening of Natural Products for Acetylcholinesterase Activity 197
5.2.4 Miniaturization of Electrospray MS Assays 198
5.2.4.1 Chip-based Electrospray MS Assays 198
5.2.4.2 Chip Performance 199
5.2.4.3 Sensitivity of the Chip-based MS Screening System 200
5.3 Continuous-flow Ligand Binding Assays Based on Mass Spectrometry 200
5.3.1 Assay Principle 200
5.3.2 Optimization of MS Conditions 201
5.3.3 On-line Continuous-flow Biochemical Interaction 202
5.3.4 Monitoring Bioactive Compounds 204
5.3.5 Antibody--Antigen Interactions 205
5.3.6 Continuous-flow Multi-protein Binding Assays Using Electrospray MS 205
5.4 MS Assay Based on Dissociation of Isolated Protein--Ligand Complexes 207
5.4.1 Assay Set-up 207
5.4.2 Flow Injection Label-free MS Assay 209
5.4.3 Flow Injection Label-free MS Assay Screening of Natural Extracts 211
5.5 Future Prospects 211
References 213
6 Frontal Affinity Chromatography -- Mass Spectrometry for Ligand Discovery and Characterization
Nora Chan, Darren Lewis, Michele Kelly, Ella S.M. Ng, and David C. Schriemer 		217
6.1 Introduction 217
6.1.1 The Basic Frontal Method 218
6.1.2 FAC -- Basic Theory 220
6.1.3 FAC Advantages 221
6.1.4 FAC Disadvantages 223
6.2 Enabling FAC with MS Detection 224
6.2.1 Direct FAC-MS Methods for Compound Binding Data 224
6.2.2 Direct Method for Discovering and Ranking Multiple Ligands 226
6.2.3 Indirect Methods 232
6.3 System Advancements -- Fluidics, Immobilization, Detection 235
6.3.1 Column 235
6.3.2 System 239
6.3.3 Breakthrough Curve Detection and Data Analysis 241
6.4 Select Applications 242
6.5 Summary and Evaluation 243
References 244
7 MS Binding Assays -- An Alternative to Radioligand Binding
Georg Höfner, Christine Zepperitz, and Klaus T. Wanner 		247
7.1 Introduction 247
7.2 Radioligand Binding Assays 248
7.2.1 General Principle 248
7.2.1.1 Saturation Assays 248
7.2.1.2 Competition Assays 249
7.2.1.3 Kinetic Assays 250
7.2.2 Application 251
7.2.3 Disadvantages and Alternatives 252
7.3 MS Binding Assays 254
7.3.1 MS Binding Assays Quantifying the Nonbound Marker 255
7.3.1.1 Competition Assays for D1 and D2 Dopamine Receptors 257
7.3.1.2 Library Screening and Competition Assays for -Opioid Receptors 263
7.3.2 MS Binding Assays Quantifying the Bound Marker 267
7.3.2.1 Saturation Assays for mGAT1 268
7.3.2.2 Competition Assays for mGAT1 272
7.3.2.3 Kinetic Assays for mGAT1 272
7.4 Summary and Perspectives 276
References 278
8 Laser Desorption Assays -- MALDI-MS, DIOS-MS, and SAMDI-MS
Martin Vogel, Andy Scheffer, André Liesener, and Uwe Karst 		285
8.1 MALDI-MS Assays 285
8.1.1 Principles of MALDI 285
8.1.2 Application of MALDI-MS in Bioanalysis 287
8.2 DIOS: Desorption/Ionization on Silicon 289
8.2.1 Principles of DIOS 289
8.2.2 Application of DIOS in Bioanalysis 292
8.3 SAMDI: Self-assembled Monolayers for MALDI-MS 295
8.3.1 Principles of SAMDI-MS 295
8.3.2 Application of SAMDI in Bioanalysis 297
8.4 Conclusion 299
References 300
III Studying target-ligand interactions analyzing intact target-ligand complexes by MS 303
9 Tethering: Fragment-based Drug Discovery by Mass Spectrometry
Mark T. Cancilla and Daniel A. Erlanson 		305
9.1 Introduction 305
9.2 Reduction to Practice 307
9.2.1 Technique 307
9.2.2 Advantages 310
9.3 Finding Fragments: Thymidylate Synthase Proof of Principle 310
9.4 Finding and Linking Fragments in One Step: Tethering with Extenders 312
9.4.1 Caspase-3 312
9.4.2 Caspase-1 316
9.5 Conclusions 316
References 318
10 Interrogation of Noncovalent Complexes by ESI-MS: A Powerful Platform for High Throughput Drug Discovery
Steven A. Hofstadler and Kristin A. Sannes-Lowery 		321
10.1 Analysis of Noncovalent Complexes by ESI-MS 321
10.1.1 Solution Conditions 321
10.1.2 Proteins 322
10.1.3 Oligonucleotides 323
10.2 Multitarget Affinity/Specificity Screening 328
10.3 Multitarget Affinity/Specificity Screening in a High Throughput Format 329
10.4 Affinity/Specificity 330
10.5 SAR by MS 332
10.6 Future Directions 333
References 335
IV Studying target-ligand interactions analyzing the target binding site by MS 339
11 Quantification of Protein--Ligand Interactions in Solution by Hydrogen/Deuterium Exchange (PLIMSTEX)
Mei M. Zhu, David Hambly, and Michael L. Gross 		341
11.1 Introduction 341
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 (Di) 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 Ki, Stoichiometry, and Protection (Di) 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)
Yoshitomo Hamuro, Stephen J. Coales, and Virgil L. Woods Jr 		377
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 PPAR{g} 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
Walter A. Korfmacher 		401
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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