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 3

Gérard Hopfgartner

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 65

Marshall M. Siegel

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 Nonvolatile 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 121

Allen Annis, Cheng-Chi Chuang, and Naim Nazef

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 157

Timothy E. Cloutier and Kenneth M. Comess

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 185

Hubertus Irth

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