A Personal Foreword ........................................ XIII
Preface ...................................................... XV
List of Contributors ....................................... XVII
I Introduction .................................................. 1
1 Administrative Optimization of Proteomics Networks for Drug
Development ................................................... 3
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 Exchange ...... 19
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 Technologies ...................................... 31
3 Difference Cel Electrophoresis (DICE): the Next Generation
of Two-Dimensional Gel Electrophoresis for Clinical
Research ..................................................... 33
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 Approaches ........................................... 57
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 Protein-Protein
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
Research .................................................... 113
Michael Schroder, 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 .................... 216
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 Field ..................... 137
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 Interactions ........................................ 159
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 Alpha Screen as a Pharmaceutical Screening Tool ......... 167
8.7 Conclusions ............................................. 170
References .................................................. 171
9 Molecular Networks in Morphologically Intact Cells and
Tissue-Challenge for Biology and Drug Development ........... 173
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 Applications .............................................. 185
10 From Target to Lead Synthesis .............................. 187
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 ВIAcore 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
Research ................................................... 209
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 Serology ........ 223
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 Proteomics ...... 241
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
Dementia ................................................... 259
Jens Wiltfang and Piotr Lewczuk
14.1 Introduction .......................................... 259
14.2 Neurochemical Markers of Alzheimer's Disease .......... 260
14.2.1 β-Amyloid Precursor Protein (β-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 Alzheimer's 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 Alzheimer's Disease .......................... 279
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 Proteomics ......................................... 299
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 PКС 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 Market .............................................. 319
17 Innovation Processes ....................................... 321
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
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