High-Throughput Mass Spectrometry in Drug Discovery

Chang Liu, Ph.D., is a Staff Research Scientist at SCIEX, a global leader in the design and production of mass spectrometers. He has published extensively on using mass spectrometry in drug discovery and development. Hui Zhang, Ph.D., is the Vice President of Analytical Technologies at Entos, a biotech company focusing on Artificial Intelligence and High-Throughput Experimentation driving drug discovery. He has published widely on mass spectrometry and its pharmaceutical applications through his previous tenure at Pfizer.

List of Contributors xv

Preface xix

List of Abbreviations xxi

Section 1 Introduction 1

1 Forty-Year Evolution of High-Throughput Mass Spectrometry: A Perspective 3
Thomas R. Covey

1.1 Introduction 3

1.2 Ionization Foundations of High-Throughput Mass Spectrometry 5

1.2.1 Historical Context of the Development of LC/MS. Ionization in Vacuum or at Atmospheric Pressure? 7

1.2.2 Ambient Sample Introduction Methods (Ambient Ionization) into an API Ion Source Without LC and Their HT-MS Potential 13

1.2.3 Direct and Indirect Affinity Measurements with ESI/MS for HTS 16

1.3 High-Speed Serial Chromatographic Sample Introduction 18

1.3.1 High Flow Rate Ion Sources 19

1.3.2 Fast Serial Scheduled, Staggered Chromatographic Separations with Fast Autosamplers 22

1.3.3 High-Speed Column Stationary Phases 24

1.4 Parallel Chromatographic Sample Introduction 26

1.4.1 Overview of Multichannel Indexed Ion Sources 26

1.4.2 Fluid Indexing 27

1.4.3 Spray Aerosol Indexing 28

1.4.4 Ion Beam Indexing 28

1.4.5 Ionization Indexing 29

1.4.6 Multichannel Autosampler and Pumps 30

1.5 High Repetition Rate Lasers 32

1.6 Ion Mobility for High-Speed Gas-Phase Separations 35

1.6.1 Motivation and Commercial Options 35

1.6.2 Origins of DMS 36

1.6.3 Chemically Based Selectivity with DMS to Mimic Chromatography 37

1.7 Mass Spectrometer Sensitivity 40

1.7.1 Historical Gains and Motivation for Sensitivity Improvements 40

1.8 High-Speed Sub-Microliter Volume Sampling 42

1.8.1 Small Sample Size and Low Volume Dispensing HT-MS Technologies 42

1.8.2 Shoot N′ Dilute Nanoliter Droplets 44

1.9 Conclusions and Future Prospects 53

References 56

Section 2 LC-MS 75

2 The LeadSampler (LS-1) Sample Delivery System: Integrated Design and Features for High-Efficiency Bioanalysis 77
Brendon Kapinos and John Janiszewski

2.1 Introduction 77

2.2 Hardware and System Design 80

2.3 Software Integration 84

2.4 Enabling Emerging Techniques 90

2.5 Concluding Remarks 96

References 97

3 Evolution of Multiplexing Technology for High-Throughput LC/MS Analyses 103
Adam Latawiec

3.1 Introduction and Historical Developments 103

3.2 Developments Toward Fully Integrated Multiplexing Systems 105

3.3 Broadening Customer Options 108

3.4 Workflow and End-User Considerations 113

3.5 Conclusion 115

References 116

Section 3 ESI-MS Without Chromatographic Separation 121

4 Direct Online SPE-MS for High-Throughput Analysis in Drug Discovery 123
Andrew D. Wagner and Wilson Z. Shou

4.1 Introduction 123

4.2 History of the Development of Direct Online SPE-MS 124

4.3 Hardware Details and Data Processing 126

4.4 Instrument Performance Highlights 132

4.5 Applications 133

4.6 Others 134

4.7 Future Perspectives 135

References 135

5 Acoustic Sampling for Mass Spectrometry: Fundamentals and Applications in High-Throughput Drug Discovery 143
Chang Liu, Lucien Ghislain, Jonathan Wingfield, Sammy Datwani, and Hui Zhang

5.1 Introduction 143

5.2 Technology Overview 145

5.2.1 Ami-MS 145

5.2.2 Ade-OPI-MS 151

5.2.2.1 System Description 151

5.2.2.2 System Tuning and Assay Development 152

5.2.2.3 ADE-OPI-MS Automated Data Processing and Automation Integration 154

5.3 System Performance 154

5.3.1 AMI-MS Performance 154

5.3.2 ADE-OPI-MS Performance 160

5.4 Applications 162

5.4.1 High-Throughput Screening 162

5.4.1.1 AMI-MS for HTS 162

5.4.1.2 ADE-OPI-MS for HTS 166

5.4.2 High-Throughput ADME 168

5.4.3 In Situ Reaction Kinetics Monitoring 168

5.4.4 Bioanalysis 170

5.4.5 Compound QC 171

5.4.6 Parallel Medicinal Chemistry 172

5.4.7 High-Content Screening 173

5.5 Challenges and Limitations 175

5.6 Conclusion 176

References 177

6 Ion Mobility Spectrometry-Mass Spectrometry for High-Throughput Analysis 183
Dylan H. Ross, Aivett Bilbao, Richard D. Smith, and Xueyun Zheng

6.1 Introduction of Ion Mobility Spectrometry 183

6.2 IMS Fundamental and Experiment 184

6.2.1 Ion Mobility Theory 184

6.2.2 Collision Cross Section Measurement 186

6.2.3 A Typical IMS Experiment 186

6.3 IMS Analysis and Applications 187

6.3.1 Separation of Isomeric and Isobaric Species by IMS 187

6.3.2 High-Throughput IMS Measurements and Building a CCS Library 188

6.3.2.1 CCS Measurement of Small Molecules Using DTIMS 190

6.3.2.2 CCS Measurements of Drug Compounds Using TWIMS 193

6.3.2.3 Large-Scale CCS Databases From Prediction Approaches 195

6.3.3 LC-IMS-MS Analysis 195

6.3.4 High-Throughput Analysis Using Rapidfire SPE-IMS-MS 196

6.3.5 Software Tools for IMS Data Analysis 199

6.4 High-Resolution SLIM-IMS Developments 200

6.5 Conclusions 204

References 205

7 Differential Mobility Spectrometry and Its Application to High-Throughput Analysis 215
Bradley B. Schneider, Leigh Bedford, Chang Liu, Eva Duchoslav, Yang Kang, Subhasish Purkayastha, Aaron Stella, and Thomas R. Covey

7.1 Introduction 215

7.2 Separation Speed 216

7.2.1 Classical Low Field Ion Mobility 216

7.2.2 Differential Mobility Spectrometry 217

7.2.2.1 FAIMS 218

7.2.2.2 DMS 219

7.3 Separation Selectivity 220

7.3.1 Classical Low Field Ion Mobility 220

7.3.2 Differential Mobility Spectrometry 220

7.3.2.1 FAIMS 220

7.3.2.2 DMS 221

7.4 Ultrahigh-Throughput System with DMS 226

7.4.1 AEMS Data 231

7.4.2 DMS Sensitivity (Ion Transmission) 237

7.4.3 Examples of AEMS Analyses with DMS 240

7.4.3.1 Example 1. DMS to Eliminate Interferences from Isobaric Species 240

7.4.3.2 Example 2. DMS to Eliminate Interferences for Species that are Not Nominally Isobaric 244

7.4.3.3 Example 3. DMS to Eliminate Unknown Interferences from Species Endogenous to the Solvent Matrix 250

7.4.4 DMS Tuning as a Component of the High-Throughput Workflow 252

7.4.5 Automation of the Tuning Process 253

7.5 Conclusions 258

7.A Chemical Structures 259

References 262

Section 4 Special Sample Arrangement 267

8 Off-Line Affinity Selection Mass Spectrometry and Its Application in Lead Discovery 269
Christopher F. Stratton, Lawrence M. Szewczuk, and Juncai Meng

8.1 Introduction to Off-Line Affinity Selection Mass Spectrometry 269

8.2 Selected Off-Line Affinity Selection Technologies and Its Application in Lead Discovery 270

8.2.1 Membrane Ultrafiltration-Based Affinity Selection 270

8.2.1.1 Introduction of Membrane Ultrafiltration-Based ASMS 270

8.2.1.2 Application of Membrane Ultrafiltration-Based ASMS in Lead Discovery 271

8.2.1.3 Pulse Ultrafiltration-Based ASMS Technology 273

8.2.1.4 Affinity Rank-Ordering Using Pulse Ultrafiltration-Based ASMS 273

8.2.1.5 Advantages and Disadvantages of Membrane Ultrafiltration-Based ASMS 275

8.2.2 Plate-Based Size Exclusion Chromatography 275

8.2.2.1 Introduction of SpeedScreen: A Plate-Based SEC ASMS Technology 275

8.2.2.2 Application of SpeedScreen in Lead Discovery 277

8.2.2.3 Advantages and Considerations of SpeedScreen 278

8.2.3 Bead-Based Affinity Selection 281

8.2.3.1 Introduction to Bead-Based Affinity Selection 281

8.2.3.2 Application and Discussion of Bead-Based Affinity Selection in Lead Discovery 282

8.2.4 Self-Assembled Monolayers and Matrix-Assisted Laser Desorption Ionization (SAMDI) 283

8.2.4.1 Introduction to SAMDI Technology 283

8.2.4.2 Discussion and Proof-of-Concept of SAMDI Technology for Off-Line ASMS 286

8.2.5 Ultracentrifugation Affinity Selection 286

8.2.5.1 Introduction to Ultracentrifugation Affinity Selection 286

8.2.5.2 Discussion and Proof-of-Concept of Ultracentrifugation Affinity Selection for Off-line ASMS 288

8.3 Future Perspectives 291

References 292

9 Online Affinity Selection Mass Spectrometry 297
Hui Zhang and Juncai Meng

9.1 Introduction of Online Affinity Selection-Mass Spectrometry 297

9.2 Online ASMS Fundamental 299

9.3 Instrument Hardware and Software Consideration 300

9.3.1 SEC Selection, Fast Separation, and Temperature 300

9.3.2 MS: Low Resolution and High Resolution 302

9.3.3 Software: Key Features, False Positives, and False Negatives 303

9.3.4 Compound Libraries and Compression Level 305

9.4 Type of Assays Using ASMS 306

9.4.1 Target Identification and Validation 306

9.4.2 Hits ID from Combinatorial Libraries or Compound Collections 308

9.4.3 Hits Characterization and Leads Optimization 308

9.5 Applications Examples and New Modalities of ASMS for Drug Discovery 311

9.6 Future Perspectives 312

References 313

10 Native Mass Spectrometry in Drug Discovery and Development 317
Mengxuan Jia, Jianzhong Wen, Olivier Mozziconacci, and Elizabeth Pierson

10.1 Introduction 317

10.1.1 The Significance of Non-Covalent Protein Complexes in Biology 317

10.1.2 Advantages and Disadvantages of Conventional Structural Analytical Techniques 318

10.2 Fundamentals of Native MS 320

10.2.1 Principles of Native Electrospray Ionization 320

10.2.2 Specific Sample Preparation to Preserve Non-Covalent Interactions and Be Compatible with ESI-MS Analysis 321

10.3 Instrumentation 323

10.3.1 Nano-ESI and ESI 323

10.3.2 Inline Desalting and Separations Coupled to Native Mass Spectrometry 323

10.3.2.1 Inline SEC and Desalting 324

10.3.2.2 Inline IEX 325

10.3.2.3 Inline HIC 325

10.3.2.4 Inline 2D LC 326

10.3.2.5 Compatibility with nESI 326

10.3.3 High-Throughput Native Mass Spectrometry 327

10.3.4 Mass Analyzers 329

10.3.5 Data Processing 329

10.3.5.1 Contrasts Between Non-Native and Native MS Data Processing and Interpretation 329

10.3.5.2 Software for Native MS 330

10.4 Application Highlights 330

10.4.1 Using Native MS to Develop Stable Protein Formulations 332

10.4.2 Native MS to Understand Drug/Target Interaction 334

10.4.3 Native Mass Spectrometry and Tractable Protein–Protein Interactions for Drug Discovery 335

10.4.4 Structural Stability Using Collision-Induced Unfolding 336

10.4.5 Vaccines and Virus Proteins Using CDMS 336

10.5 Conclusions and Future Directions 337

References 337

Section 5 Other Ambient Ionization Other than ESI 347

11 Laser Diode Thermal Desorption-Mass Spectrometry (LDTD-MS): Fundamentals and Applications of Sub-Second Analysis in Drug Discovery Environment 349
Pierre Picard, Sylvain Letarte, Jonathan Rochon, and Réal E. Paquin

11.1 A Historical Perspective of the LDTD 349

11.2 Instrumentation 351

11.2.1 LDTD Process 351

11.2.2 Sample Holder Design 352

11.2.3 Vapor Extraction Nozzle 353

11.3 Theoretical Background 354

11.3.1 Thermal Process 354

11.3.2 Gas Dynamics 358

11.3.3 Ionization 359

11.4 Sample Preparation 362

11.4.1 Motivations 362

11.4.2 General Guidelines 362

11.4.2.1 Compound Detection Background 363

11.4.2.2 Details on Ionic Saturation 364

11.4.2.3 Consideration for Biological Matrices 367

11.5 Applications 370

11.5.1 CYP Inhibition Analysis 371

11.5.2 Permeability 373

11.5.3 Protein Binding 378

11.5.4 Pharmacokinetic 378

11.5.5 Preparation Tips 382

11.6 Conclusion 384

11.6.1 Use and Merits of the Technology 384

11.6.2 Limitations 385

11.6.3 Perspectives 386

References 387

12 Accelerating Drug Discovery with Ultrahigh-Throughput MALDI-TOF MS 393
Sergei Dikler

12.1 Introduction 393

12.2 uHT-MALDI MS of Assays and Chemical Reactions 396

12.2.1 HT-MALDI of Enzymatic Assays 396

12.2.2 Screening Chemical Reactions Using uHT-MALDI 401

12.2.3 uHT-MALDI of Cell-Based Assays 404

12.2.4 uHT-MALDI of Other Types of Assays and Libraries 406

12.3 Bead-Based Workflows 408

12.4 Using Functionalized, Modified, and Microarrayed MALDI Plates for HT-MALDI 411

12.5 Summary and Future Trends 413

Acknowledgment 414

References 414

13 Development and Applications of DESI-MS in Drug Discovery 423
Wenpeng Zhang

13.1 Introduction 423

13.2 Development of DESI and Related Ambient Ionization Methods 424

13.3 Applications in Drug Discovery 427

13.3.1 Pharmaceutical Analysis and Therapeutic Drug Monitoring 427

13.3.2 Analysis of Drugs in Natural Products 428

13.3.3 DESI-Based Mass Spectrometry Imaging 430

13.3.4 Detection of Drug–Protein Interactions 435

13.3.5 High-Throughput Experimentation 438

13.3.6 High-Throughput Screening 439

13.4 Conclusions and Future Outlook 440

References 442

Section 6 Conclusion 453

14 The Impact of HT-MS to Date and Its Potential to Shape the Future of Metrics-Based Experimentation and Analysis 455
Matthew D. Troutman

14.1 Defining High-Throughput Mass Spectrometry (HT-MS) 456

14.2 HT-MS: Impact to Date 457

14.3 Considering How HT-MS Will Shape the Future of Drug Discovery 458

References 462

Index 467