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

High-Throughput Mass Spectrometry in Drug Discovery
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