Info

Introduction 185

Continuous-flow Enzyme Assays Based on Mass Spectrometry 186

5.2.1 Assay Principle 286

5.2.2 ESI-MS Assay of Cathepsin B 288

5.2.2.1 MS Assay Development for Cathepsin B 288

5.2.2.2 Compatibility of Cathepsin B Assay with MS Detection 288

5.2.2.3 On-line Coupling of MS-based Cathepsin B Assay to HPLC 290

5.2.2.4 Screening of Natural Products for Cathepsin B Activity 292

5.2.3 ESI-MS Assay of Acetylcholinesterase 294

5.2.3.1 MS Assay Development for Acetylcholinesterase 294

5.2.3.2 Assay Validation and Stability 297

5.2.3.3 Screening of Natural Products for Acetylcholinesterase Activity 297

5.2.4 Miniaturization of Electrospray MS Assays 298

5.2.4.1 Chip-based Electrospray MS Assays 298

5.2.4.2 Chip Performance 299

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 202

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 222

5.5 Future Prospects 222 References 223

6 Frontal Affinity Chromatography - Mass Spectrometry for Ligand Discovery and Characterization 227

Nora Chan, Darren Lewis, Michele Kelly, Ella S.M. Ng, and David C. Schriemer

6.1 Introduction 227

6.1.1 The Basic Frontal Method 228

6.1.2 FAC - Basic Theory 220

6.1.3 FAC Advantages 222

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 247

Georg Hofner, Christine Zepperitz, and Klaus T. Wanner

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 m-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 285

Martin Vogel, Andy Scheffer, Andre Liesener, and Uwe Karst

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 305

Mark T. Cancilla and Daniel A. Erlanson

9.1 Introduction 305

9.2 Reduction to Practice 307

9.2.1 Technique 307

9.2.2 Advantages 320

9.3 Finding Fragments: Thymidylate Synthase Proof of Principle 320

9.4 Finding and Linking Fragments in One Step: Tethering with Extenders 322

9.4.1 Caspase-3 322

9.4.2 Caspase-1 326

9.5 Conclusions 326 References 328

10 Interrogation of Noncovalent Complexes by ESI-MS: A Powerful Platform for High Throughput Drug Discovery 322

Steven A. Hofstadler and Kristin A. Sannes-Lowery

10.1 Analysis of Noncovalent Complexes by ESI-MS 322

10.1.1 Solution Conditions 322

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

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