Proteomics in Drug Research (E-Book) von Michael Hamacher

A Personal Foreword xiii

Preface xv

List of Contributors xvii

I Introduction1

1 Administrative Optimization of Proteomics Networks for Drug Development3
André van Hall and Michael Hamacher

1.1 Introduction 3

1.2 Tasks and Aims of Administration 4

1.3 Networking 6

1.4 Evaluation of Biomarkers 7

1.5 A Network for Proteomics in Drug Development 9

1.6 Realization of Administrative Networking: the Brain Proteome Projects 10

1.6.1 National Genome Research Network: the Human Brain Proteome Project 11

1.6.2 Human Proteome Organisation: the Brain Proteome Project 14

1.6.2.1 The Pilot Phase 15

References 17

2 Proteomic Data Standardization, Deposition and Exchange19
Sandra Orchard, Henning Hermjakob, Manuela Pruess, and Rolf Apweiler

2.1 Introduction 19

2.2 Protein Analysis Tools 21

2.2.1 UniProt 21

2.2.2 InterPro 22

2.2.3 Proteome Analysis 22

2.2.4 International Protein Index (IPI) 23

2.2.5 Reactome 23

2.3 Data Storage and Retrieval 23

2.4 The Proteome Standards Initiative 24

2.5 General Proteomics Standards (GPS) 24

2.6 Mass Spectrometry 25

2.7 Molecular Interactions 27

2.8 Summary 28

References 28

II Proteomic Technologies31

3 Difference Gel Electrophoresis (DIGE): the Next Generation of Two-Dimensional Gel Electrophoresis for Clinical Research33
Barbara Sitek, Burghardt Scheibe, Klaus Jung, Alexander Schramm and Kai Stühler

3.1 Introduction 34

3.2 Difference Gel Electrophoresis: Next Generation of Protein Detection in 2-DE 36

3.2.1 Application of CyDye DIGE Minimal Fluors (Minimal Labeling with CyDye DIGE Minimal Fluors) 38

3.2.1.1 General Procedure 38

3.2.1.2 Example of Use: Identification of Kinetic Proteome Changes upon Ligand Activation of Trk-Receptors 39

3.2.2 Application of Saturation Labeling with CyDye DIGE Saturation Fluors 44

3.2.2.1 General Procedure 44

3.2.2.2 Example of Use: Analysis of 1000 Microdissected Cells from PanIN Grades for the Identification of a New Molecular Tumor Marker Using CyDye DIGE Saturation Fluors 45

3.2.3 Statistical Aspects of Applying DIGE Proteome Analysis 47

3.2.3.1 Calibration and Normalization of Protein Expression Data 48

3.2.3.2 Detection of Differentially Expressed Proteins 50

3.2.3.3 Sample Size Determination 51

3.2.3.4 Further Applications 52

References 52

4 Biological Mass Spectrometry: Basics and Drug Discovery Related Approaches57
Bettina Warscheid

4.1 Introduction 57

4.2 Ionization Principles 58

4.2.1 Matrix-Assisted Laser Desorption/Ionization (MALDI) 58

4.2.2 Electrospray Ionization 60

4.3 Mass Spectrometric Instrumentation 62

4.4 Protein Identification Strategies 65

4.5 Quantitative Mass Spectrometry for Comparative and Functional Proteomics 67

4.6 Metabolic Labeling Approaches 69

4.6.1 15N Labeling 70

4.6.2 Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) 71

4.7 Chemical Labeling Approaches 73

4.7.1 Chemical Isotope Labeling at the Protein Level 73

4.7.2 Stable Isotope Labeling at the Peptide Level 75

4.8 Quantitative MS for Deciphering ProteinProtein Interactions 78

4.9 Conclusions 80

References 81

5 Multidimensional Column Liquid Chromatography (LC) in Proteomics Where are We Now?89
Egidijus Machtejevas, Klaus K. Unger and Reinhard Ditz

5.1 Introduction 90

5.2 Why Do We Need MD-LC/MS Methods? 91

5.3 Basic Aspects of Developing a MD-LC/MS Method 92

5.3.1 General 92

5.3.2 Issues to be Considered 93

5.3.3 Sample Clean-up 94

5.3.4 Choice of Phase Systems in MD-LC 94

5.3.5 Operational Aspects 97

5.3.6 State-of-the-Art Digestion Strategy Included 98

5.3.6.1 Multidimensional LC MS Approaches 98

5.4 Applications of MD-LC Separation in Proteomics a Brief Survey 100

5.5 Sample Clean-Up: Ways to Overcome the Bottleneck in Proteome Analysis 104

5.6 Summary 109

References 110

6 Peptidomics Technologies and Applications in Drug Research113
Michael Schrader, Petra Budde, Horst Rose, Norbert Lamping, Peter Schulz-Knappe and Hans-Dieter Zucht

6.1 Introduction 114

6.2 Peptides in Drug Research 114

6.2.1 History of Peptide Research 114

6.2.2 Brief Biochemistry of Peptides 116

6.2.3 Peptides as Drugs 117

6.2.4 Peptides as Biomarkers 118

6.2.5 Clinical Peptidomics 118

6.3 Development of Peptidomics Technologies 120

6.3.1 Evolution of Peptide Analytical Methods 120

6.3.2 Peptidomic Profiling 121

6.3.3 Top-Down Identification of Endogenous Peptides 123

6.4 Applications of Differential Display Peptidomics 124

6.4.1 Peptidomics in Drug Development 124

6.4.2 Peptidomics Applied to in vivo Models 127

6.5 Outlook 129

References 130

7 Protein Biochips in the Proteomic Field137
Angelika Lücking and Dolores J. Cahill

7.1 Introduction 137

7.2 Technological Aspects 139

7.2.1 Protein Immobilization and Surface Chemistry 139

7.2.2 Transfer and Detection of Proteins 141

7.2.3 Chip Content 142

7.3 Applications of Protein Biochips 144

7.4 Contribution to Pharmaceutical Research and Development 150

References 151

8 Current Developments for the In Vitro Characterization of Protein Interactions159
Daniela Moll, Bastian Zimmermann, Frank Gesellchen and Friedrich W. Herberg

8.1 Introduction 160

8.2 The Model System: cAMP-Dependent Protein Kinase 161

8.3 Real-time Monitoring of Interactions Using SPR Biosensors 161

8.4 ITC in Drug Design 163

8.5 Fluorescence Polarization, a Tool for High-Throughput Screening 165

8.6 AlphaScreen as a Pharmaceutical Screening Tool 167

8.7 Conclusions 170

References 171

9 Molecular Networks in Morphologically Intact Cells and TissueChallenge for Biology and Drug Development173
Walter Schubert, Manuela Friedenberger and Marcus Bode

9.1 Introduction 173

9.2 A Metaphor of the Cell 174

9.3 Mapping Molecular Networks as Patterns: Theoretical Considerations 176

9.4 Imaging Cycler Robots 177

9.5 Formalization of Network Motifs as Geometric Objects 179

9.6 Gain of Functional Information: Perspectives for Drug Development 182

References 182

III Applications185

10 From Target to Lead Synthesis187
Stefan Müllner, Holger Stark, Päivi Niskanen, Erich Eigenbrodt, Sybille Mazurek and Hugo Fasold

10.1 Introduction 187

10.2 Materials and Methods 190

10.2.1 Cells and Culture Conditions 190

10.2.2 In Vitro Activity Testing 190

10.2.3 Affinity Chromatography 190

10.2.4 Electrophoresis and Protein Identification 191

10.2.5 BIAcore Analysis 191

10.2.6 Synthesis of Acyl Cyanides 192

10.2.6.1 Methyl 5-cyano-5-oxopentanoate 192

10.2.6.2 Methyl 6-cyano-6-oxohexanoate 193

10.2.6.3 Methyl-5-cyano-3-methyl-5-oxopentanoate 193

10.3 Results 193

10.4 Discussion 201

References 203

11 Differential Phosphoproteome Analysis in Medical Research209
Elke Butt and Katrin Marcus

11.1 Introduction 210

11.2 Phosphoproteomics of Human Platelets 211

11.2.1 Cortactin 213

11.2.2 Myosin Regulatory Light Chain 213

11.2.3 Protein Disulfide Isomerase 214

11.3 Identification of cAMP- and cGMP-Dependent Protein Kinase Substrates in Human Platelets 216

11.4 Identification of a New Therapeutic Target for Anti-Inflammatory Therapy by Analyzing Differences in the Phosphoproteome of Wild Type and Knock Out Mice 218

11.5 Concluding Remarks and Outlook 219

References 220

12 Biomarker Discovery in Renal Cell Carcinoma Applying Proteome-Based Studies in Combination with Serology223
Barbara Seliger and Roland Kellner

12.1 Introduction 224

12.1.1 Renal Cell Carcinoma 224

12.2 Rational Approaches Used for Biomarker Discovery 225

12.3 Advantages of Different Proteome-Based Technologies for the Identification of Biomarkers 226

12.4 Type of Biomarker 228

12.5 Proteome Analysis of Renal Cell Carcinoma Cell Lines and Biopsies 229

12.6 Validation of Differentially Expressed Proteins 234

12.7 Conclusions 235

References 235

13 Studies of Drug Resistance Using Organelle Proteomics241
Catherine Fenselau and Zongming Fu

13.1 Introduction 242

13.1.1 The Clinical Problem and the Proteomics Response 242

13.2 Objectives and Experimental Design 243

13.2.1 The Cell Lines 243

13.2.2 Organelle Isolation 244

13.2.2.1 Criteria for Isolation 244

13.2.2.2 Plasma Membrane Isolation 245

13.2.3 Protein Fractionation and Identification 247

13.2.4 Quantitative Comparisons of Protein Abundances 249

13.3 Changes in Plasma Membrane and Nuclear Proteins in MCF-7 Cells Resistant to Mitoxantrone 252

References 254

14 Clinical Neuroproteomics of Human Body Fluids: CSF and Blood Assays for Early and Differential Diagnosis of Dementia259
Jens Wiltfang and Piotr Lewczuk

14.1 Introduction 259

14.2 Neurochemical Markers of Alzheimers Disease 260

14.2.1 -Amyloid Precursor Protein (-APP): Metabolism and Impact on AD Diagnosis 261

14.2.2 Tau Protein and its Phosphorylated Forms 263

14.2.2.1 Hyperphosphorylation of Tau as a Pathological Event 264

14.2.2.2 Phosphorylated Tau in CSF as a Biomarker of Alzheimers Disease 265

14.2.3 Apolipoprotein E (ApoE) Genotype 266

14.2.4 Other Possible Factors 267

14.2.5 Combined Analysis of CSF Parameters 267

14.2.6 Perspectives: Novel Techniques to Search for AD Biomarkers Mass Spectrometry (MS), Differential Gel Electrophoresis (DIGE), and Multiplexing 270

14.3 Conclusions 271

References 272

15 Proteomics in Alzheimers Disease279
Michael Fountoulakis, Sophia Kossida and Gert Lubec

15.1 Introduction 279

15.2 Proteomic Analysis 280

15.2.1 Sample Preparation 280

15.2.2 Two-Dimensional Electrophoresis 282

15.2.3 Protein Quantification 282

15.2.4 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectroscopy 283

15.3 Proteins with Deranged Levels and Modifications in AD 284

15.3.1 Synaptosomal Proteins 290

15.3.2 Guidance Proteins 291

15.3.3 Signal Transduction Proteins 291

15.3.4 Oxidized Proteins 292

15.3.5 Heat Shock Proteins 293

15.3.6 Proteins Enriched in Amyloid Plaques 293

15.4 Limitations 294

References 294

16 Cardiac Proteomics299
Emma McGregor and Michael J. Dunn

16.1 Heart Proteomics 300

16.1.1 Heart 2-D Protein Databases 300

16.1.2 Dilated Cardiomyopathy 300

16.1.3 Animal Models of Heart Disease 301

16.1.4 Subproteomics of the Heart 302

16.1.4.1 Mitochondria 302

16.1.4.2 PKC Signal Transduction Pathways 304

16.1.5 Proteomics of Cultured Cardiac Myocytes 305

16.1.6 Proteomic Characterization of Cardiac Antigens in Heart Disease and Transplantation 306

16.1.7 Markers of Acute Allograft Rejection 307

16.2 Vessel Proteomics 307

16.2.1 Proteomics of Intact Vessels 307

16.2.2 Proteomics of Isolated Vessel Cells 308

16.2.3 Laser Capture Microdissection 311

16.3 Concluding Remarks 312

References 312

IV To the Market319

17 Innovation Processes321
Sven Rüger

17.1 Introduction 321

17.2 Innovation Process Criteria 322

17.3 The Concept 322

17.4 Market Attractiveness 323

17.5 Competitive Market Position 323

17.6 Competitive Technology Position 324

17.7 Strengthen the Fit 325

17.8 Reward 325

17.9 Risk 325

17.10 Innovation Process Deliverables for each Stage 326

17.11 Stage Gate-Like Process 326

17.11.1 Designation as an Evaluation Project (EvP) 327

17.11.2 Advancement to Exploratory Project (EP) 329

17.11.3 For Advancement to Progressed Project (PP) 331

17.11.4 Advancement to Market Preparation 334

17.12 Conclusion 335

Subject Index 337

From skillful handling of the wide range of technologies to successful applications in drug discovery -- this handbook has all the information professional proteomics users need.
Edited by experts working at one of the hot spots in European proteomic research, the numerous contributions by experts from the pharmaceutical industry and public proteomics consortia to provide the necessary perspective on current trends and developments in this exciting field.
Following an introductory chapter, the book moves on to proteomic technologies, such as protein biochips, protein-protein interactions, and proteome analysis in situ. The section on applications includes bioinformatics, Alzheimer's disease, neuroproteomics, plasma and T-cell proteomics, differential phosphoproteome analysis and biomarkers, as well as pharmacogenomics.
Invaluable reading for medicinal and pharmaceutical chemists, gene technologists, molecular biologists, and those working in the pharmaceutical industry.

All six editors are Researchers at the Medical Proteom-Center hosted by the University of Bochum (Germany). This international research center was established in 2002 under the leadership of Helmut E. Meyer, a co-founder of the Protagen AG. Professor Meyer is also initiator and coordinator of the Human Brain Proteome Project within the German National Genome Research Net (NGFN) as well as of the Brain Proteome Project within the Human Proteome Organisation (HUPO BPP).