Proteomics

Part I: Technologies & Markets

By
Prof. K. K. Jain
MD, FRACS, FFPM

Jain PharmaBiotech
Basel, Switzerland

July 2012

A Jain PharmaBiotech Report

AUTHOR'S

BIOGRAPHY

Professor K. K. Jain is a neurologist/neurosurgeon by training and has been working in

the biotechnology/biopharmaceuticals industry for several years. He received graduate
training in both Europe and USA, has held academic positions in several countries, and is
a Fellow of the Faculty of Pharmaceutical Medicine of the Royal College of Physicians of
UK. Currently, he is a consultant and CEO at Jain PharmaBiotech.
Prof. Jain is the author of 440 publications including 21 books (4 as editor) and 50 special
reports which have covered important areas in biotechnology, gene therapy and
biopharmaceuticals. He has also written a textbook of gene therapy which is the first book
on this subject to be translated into the Chinese language. A book on gene therapy
companies was published in 2000 by John Wiley & Sons. Both of these are now
incorporated in a special report on gene therapy published in 2004 by Jain
PharmaBiotech with the latest edition in 2011. His latest books include "Handbook of
Nanomedicine" (Humana/Springer 2008; Chinese edition, Peking University Press 2011,
2nd ed Springer 2012), Textbook of Personalized Medicine (Springer 2009), "Handbook of
Biomarkers" (Springer 2010), "Handbook of Neuroprotection" (Springer 2011),
Applications of Biotechnology in Cardiovascular Therapeutics (Springer 2011), and
Applications of Biotechnology in Neurology (Springer 2012).

ABOUT

THIS

REPORT

The proteomics report started as an expansion of the chapter on proteomics in Prof.

Jain's report on "The Impact of Genomics on Drug Discovery and Development"
published by Decision Resources Inc in 1999 and has been constantly updated since its
publication as a separate report in 2000 by Jain PharmaBiotech. It has been rewritten
several times since then.

July 2012 (first edition published in 2000)

Copyright 2012 by
Jain PharmaBiotech
Blsiring 7
CH-4057 Basel
Switzerland
Tel & Fax:
Email:
Web site:

+4161-6924461
info@pharmabiotech.ch
http://pharmabiotech.ch/

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, or otherwise without the prior written
permission of the Publisher. This report may not be lent, resold or otherwise traded in any manner without the
consent of the Publisher. While all reasonable steps have been taken to ensure the accuracy of the information
presented, the Publisher cannot accept responsibility for inadvertent errors or omissions.

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TABLE

OF

CONTENTS

0. Executive Summary .............................................................................. 17

1. Basics of Proteomics ............................................................................. 19
Introduction....................................................................................................................... 19
History ............................................................................................................................... 19
Nucleic acids, genes and proteins ...................................................................................... 20
Genome .......................................................................................................................... 20
DNA ............................................................................................................................... 21
RNA ............................................................................................................................... 21
MicroRNAs .................................................................................................................. 21
Decoding of mRNA by the ribosome ............................................................................... 22
Genes............................................................................................................................. 23
Alternative splicing ........................................................................................................... 23
Transcription ................................................................................................................... 24
Gene regulation ............................................................................................................... 24
Gene expression .............................................................................................................. 25
Chromatin ....................................................................................................................... 25
Golgi complex .................................................................................................................. 26
Proteins .......................................................................................................................... 26
Spliceosome ............................................................................................................... 27
Functions of proteins ................................................................................................... 27
Inter-relationship of protein, mRNA and DNA .................................................................. 28
Proteomics ......................................................................................................................... 29
Mitochondrial proteome .................................................................................................... 30
S-nitrosoproteins in mitochondria.................................................................................. 30
Proteomics and genomics .................................................................................................. 31
Classification of proteomics ............................................................................................... 33
Levels of functional genomics and various "omics" ............................................................... 33
Glycoproteomics .............................................................................................................. 34
Transcriptomics ............................................................................................................... 34
Metabolomics .................................................................................................................. 34
Cytomics......................................................................................................................... 35
Phenomics ......................................................................................................................... 35
Proteomics and systems biology ........................................................................................ 35
Functional synthetic proteins ............................................................................................. 36

2. Proteomic Technologies ........................................................................ 37

Key technologies driving proteomics ................................................................................. 37
Sample preparation ........................................................................................................... 38
New trends in sample preparation ...................................................................................... 38
Pressure Cycling Technology ............................................................................................. 39
Protein separation technologies ........................................................................................ 39
High resolution 2DGE ....................................................................................................... 39
Variations of 2D gel technology.......................................................................................... 40
Limitations of 2DGE and measures to overcome these .......................................................... 40
1-D sodium dodecyl sulfate (SDS) PAGE ............................................................................. 40
Capillary electrophoresis systems ....................................................................................... 41
Head column stacking capillary zone electrophoresis ............................................................ 41
Removal of albumin and IgG ............................................................................................. 41
Companies with protein separation technologies .................................................................. 42
Protein purification ............................................................................................................ 43
Technologies for protein purification ................................................................................... 44
Applications of protein purification ..................................................................................... 44
Protein detection ............................................................................................................... 44
Protein identification and characterization ........................................................................ 44
Mass spectrometry (MS) ................................................................................................... 44
Companies involved in mass spectrometry ..................................................................... 45
Electrospray ionization ................................................................................................. 46
Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry ....................................... 47
Cryogenic MALDI- Fourier Transform Mass Spectrometry ................................................. 48
Stable-isotope-dilution tandem mass spectrometry ......................................................... 49
HUPO Gold MS Protein Standard ................................................................................... 49
High performance liquid chromatography ............................................................................ 49
Multidimensional protein identification technology (MudPIT) .................................................. 49
Peptide mass fingerprinting ............................................................................................... 50
Combination of protein separation technologies with mass spectrometry ................................. 50
Combining capillary electrophoresis with mass spectrometry ............................................ 50

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2D PAGE and mass spectrometry .................................................................................. 50

Quantification of low abundance proteins ....................................................................... 51
SDS-PAGE .................................................................................................................. 51
Antibodies and proteomics ................................................................................................ 52
Detection of fusion proteins ............................................................................................... 52
Labeling and detection of proteins ...................................................................................... 52
Fluorescent labeling of proteins in living cells .................................................................. 53
Combination of microspheres with fluorescence .............................................................. 53
Self-labeling protein tags ............................................................................................. 53
Analysis of peptides ......................................................................................................... 54
C-terminal peptide analysis .......................................................................................... 54
Differential Peptide Display ........................................................................................... 55
Peptide analyses using NanoLC-MS ............................................................................... 55
Protein sequencing ............................................................................................................ 56
Real-time PCR for protein quantification ............................................................................ 57
Quantitative proteomics .................................................................................................... 57
MS-based quantitative proteomics...................................................................................... 57
MS and cryo-electron tomography ...................................................................................... 57
Functional proteomics: technologies for studying protein function .................................... 58
Functional genomics by mass spectrometry ......................................................................... 58
RNA-Protein fusions ........................................................................................................... 58
Designed repeat proteins ................................................................................................... 58
Application of nanbiotechnology to proteomics ................................................................. 59
Nanoproteomics ............................................................................................................... 59
Protein nanocrystallography ......................................................................................... 59
Single-molecule mass spectrometry using a nanopore ..................................................... 60
Nanoelectrospray ionization .......................................................................................... 60
Nanoparticle barcodes ...................................................................................................... 61
Biobarcode assay for proteins ....................................................................................... 61
Nanoproteomics for discovery of protein biomarkers in the blood ........................................... 62
Nanoscale protein analysis ................................................................................................ 62
Nanoscale mechanism for protein engineering ..................................................................... 63
Nanotube electronic biosensor ........................................................................................... 63
Nanotube-vesicle networks for study of membrane proteins .................................................. 64
Nanowire transistor for the detection of protein-protein interactions ....................................... 64
Qdot-nanocrystals ............................................................................................................ 64
Resonance Light Scattering technology ............................................................................... 65
Study of single membrane proteins at subnanometer resolution............................................. 65
Protein expression profiling ............................................................................................... 65
Cell-based protein assays .................................................................................................. 66
Living cell-based assays for protein function ........................................................................ 67
Companies developing cell-based protein assays .................................................................. 67
Protein function studies ..................................................................................................... 68
Transcriptionally Active PCR .............................................................................................. 68
Protein-protein interactions ............................................................................................... 68
Yeast two-hybrid system .............................................................................................. 70
Membrane one-hybrid method ...................................................................................... 71
Protein affinity chromatography .................................................................................... 71
Phage display ............................................................................................................. 71
Fluorescence Resonance Energy Transfer ....................................................................... 72
Bioluminescence Resonance Energy Transfer .................................................................. 72
Detection Enhanced Ubiquitin Split Protein Sensor technology .......................................... 72
Protein-fragment complementation system .................................................................... 73
In vivo study of protein-protein interactions ................................................................... 73
Computational prediction of interactions ........................................................................ 74
Interactome ............................................................................................................... 74
Protein-protein interactions and drug discovery .............................................................. 75
Companies with technologies for protein-protein interaction studies .................................. 75
Protein-DNA interaction .................................................................................................... 76
Determination of protein structure .................................................................................... 76
X-Ray crystallography ...................................................................................................... 77
Nuclear magnetic resonance .............................................................................................. 78
Electron spin resonance .................................................................................................... 78
Prediction of protein structure ........................................................................................... 78
Protein tomography .......................................................................................................... 79
X-ray scattering-based method for determining the structure of proteins ................................ 80
Prediction of protein function ............................................................................................ 80
Three-dimensional proteomics for determination of function .................................................. 80
An algorithm for genome-wide prediction of protein function ................................................. 81
Monitoring protein function by expression profiling ............................................................... 81
Isotope-coded affinity tag peptide labeling ....................................................................... 81

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Differential Proteomic Panning .......................................................................................... 82

Cell map proteomics .......................................................................................................... 83
Topological proteomics ...................................................................................................... 83
Organelle or subcellular proteomics .................................................................................. 84
Nucleolar proteomics ......................................................................................................... 84
Glycoproteomic technologies ............................................................................................. 85
High-sensitivity glycoprotein analysis ................................................................................. 85
Fluorescent in vivo imaging of glycoproteins ........................................................................ 85
Integrated approaches for protein characterization .......................................................... 85
Imaging mass spectrometry .............................................................................................. 86
IMS technologies ............................................................................................................. 86
Applications of IMS........................................................................................................... 86
The protein microscope ..................................................................................................... 87
Automation and robotics in proteomics ............................................................................. 87
Western blot ...................................................................................................................... 88
Limitations of WB ............................................................................................................. 88
Innovations in WB ............................................................................................................ 88
Capillary electrophoresis and WB .................................................................................. 89
Microfluidics and WB.................................................................................................... 89
Multiplexing WB .......................................................................................................... 89
Applications of Western blot .............................................................................................. 89
Research applications of Western blot ............................................................................ 90
Molecular diagnostic applications of Western blot ............................................................ 90
Companies involved in Western blotting technologies ........................................................... 90
Laser capture microdissection ........................................................................................... 91
Microdissection techniques used for proteomics ................................................................... 91
Uses of LCM in combination with proteomic technologies....................................................... 92
Concluding remarks about applications of proteomic technologies .................................... 92
Precision proteomics......................................................................................................... 93

3. Protein biochip technology ................................................................... 95

Introduction....................................................................................................................... 95
Types of protein biochips ................................................................................................... 96
ProteinChip ..................................................................................................................... 96
Applications and advantages of ProteinChip .................................................................... 97
ProteinChip Biomarker System ...................................................................................... 97
Matrix-free ProteinChip Array ....................................................................................... 98
Aptamer-based protein biochip .......................................................................................... 98
Fluorescence planar wave guide technology-based protein biochips ........................................ 99
Lab-on-a-chip for protein analysis ...................................................................................... 99
Biochips for peptide arrays .............................................................................................. 100
Microfluidic biochips for proteomics .................................................................................. 100
Protein biochips for high-throughput expression ................................................................. 101
Nucleic Acid-Programmable Protein Array ..................................................................... 101
High-density protein microarrays ................................................................................ 101
HPLC-Chip for protein identification ............................................................................. 102
Antibody microarrays................................................................................................. 102
Integration of protein array and image analysis ............................................................ 102
Tissue microarray technology for proteomics .................................................................. 103
Protein biochips in molecular diagnostics ........................................................................ 103
A force-based protein biochip .......................................................................................... 104
L1 chip and lipid immobilization ....................................................................................... 104
Multiplexed Protein Profiling on Microarrays ....................................................................... 104
Live cell microarrays ........................................................................................................ 105
ProteinArray Workstation ................................................................................................ 105
Proteome arrays .............................................................................................................. 106
The Yeast ProtoArray ...................................................................................................... 106
ProtoArray Human Protein Microarray ............................................................................ 106
TRINECTIN proteome chip ............................................................................................... 107
Peptide arrays.................................................................................................................. 107
Surface plasmon resonance technology ........................................................................... 108
Biacore's SPR ................................................................................................................ 108
FLEX CHIP ..................................................................................................................... 108
Combination of surface plasmon resonance and MALDI-TOF ................................................ 109
Protein chips/microarrays using nanotechnology ............................................................ 109
Nanoparticle protein chip ................................................................................................ 109
Protein nanobiochip ........................................................................................................ 110
Protein nanoarrays ......................................................................................................... 110
Self-assembling protein nanoarrays .................................................................................. 110
Companies involved in protein biochip/microarray technology ....................................... 111

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4. Bioinformatics in Relation to Proteomics ............................................ 115

Introduction..................................................................................................................... 115
Bioinformatic tools for proteomics ................................................................................... 115
Testing of SELDI-TOF MS Proteomic Data .......................................................................... 115
BioImagines ProteinMine ................................................................................................ 116
Bioinformatics for pharmaceutical applications of proteomics ......................................... 116
In silico search of drug targets by Biopendium ................................................................... 116
Compugen's LEADS ........................................................................................................ 117
DrugScore ..................................................................................................................... 117
Proteochemometric modeling .......................................................................................... 117
Integration of genomic and proteomic data ..................................................................... 118
Proteomic databases: creation and analysis .................................................................... 119
Introduction .................................................................................................................. 119
Proteomic databases ...................................................................................................... 119
GenProtEC ............................................................................................................... 120
Human Protein Atlas .................................................................................................. 120
Human Proteomics Initiative ....................................................................................... 121
International Protein Index ......................................................................................... 122
Proteome maps......................................................................................................... 122
Protein Structure Initiative Structural Genomics Knowledgebase ................................... 122
Protein Warehouse Database ...................................................................................... 122
Protein Data Bank ..................................................................................................... 123
Repository for raw data from proteomics MS ................................................................ 123
Universal Protein Resource ......................................................................................... 123
Protein interaction databases........................................................................................... 124
Biomolecular Interaction Network Database.................................................................. 125
ENCODE ....................................................................................................................... 125
Functional Genomics Consortium................................................................................. 125
Human Proteinpedia .................................................................................................. 126
ProteinCenter ........................................................................................................... 126
Databases of the National Center for Biotechnology Information ..................................... 126
Bioinformatics for protein identification .......................................................................... 127
Application of bioinformatics in functional proteomics .................................................... 127
Use of bioinformatics in protein sequencing .................................................................... 127
Bottom-up protein sequencing ......................................................................................... 128
Top-down protein sequencing .......................................................................................... 129
Protein structural database approach to drug design ...................................................... 129
Bioinformatics for high-throughput proteomics ............................................................... 129
Companies with bioinformatic tools for proteomics ......................................................... 130

5. Research in Proteomics ...................................................................... 132

Introduction..................................................................................................................... 132
Applications of proteomics in biological research ............................................................ 132
Identification of novel human genes by comparative proteomics .......................................... 132
Study of relationship between genes and proteins .............................................................. 133
Characterization of histone codes ..................................................................................... 133
Structural genomics or structural proteomics .................................................................. 134
Protein Structure Factory ................................................................................................ 135
Protein Structure Initiative .............................................................................................. 135
Studies on protein structure at Argonne National Laboratory ............................................... 136
Structural Genomics Consortium ...................................................................................... 136
Protein knockout.............................................................................................................. 137
Antisense approach and proteomics ................................................................................. 137
RNAi and protein knockout .............................................................................................. 137
Total knockout of cellular proteins .................................................................................... 137
Ribozymes and proteomics .............................................................................................. 138
Single molecule proteomics ............................................................................................. 138
Single-molecule photon stamping spectroscopy ................................................................. 138
Single nucleotide polymorphism determination by TOF-MS .................................................. 139
Application of proteomic technologies in systems biology ............................................... 139
Signaling pathways and proteomics .................................................................................. 139
Kinomics ....................................................................................................................... 140
Combinatorial RNAi for quantitative protein network analysis............................................... 140
Proteomics in neuroscience research ............................................................................... 140
Stem cell proteomics ....................................................................................................... 141
Comparative proteomic analysis of somatic cells, iPSCs and ESCs ........................................ 141
hESC phosphoproteome .................................................................................................. 142
Proteomic studies of mesenchymal stem cells .................................................................... 142
Proteomics of neural stem cells ........................................................................................ 143
Proteome Biology of Stem Cells Initiative .......................................................................... 143
Proteomic analysis of the cell cycle ................................................................................. 144

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Nitric oxide and proteomics ............................................................................................. 144

A proteomic method for identification of cysteine S-nitrosylation sites .................................. 144
Study of the nitroproteome ............................................................................................. 145
Study of the phosphoproteome ........................................................................................ 145
Study of the mitochondrial proteome ............................................................................... 146
Proteomic technologies for study of mitochondrial proteomics.............................................. 146
Cryptome ......................................................................................................................... 147
Study of protein transport in health and disease ............................................................. 147
Proteomics research in the academic sector .................................................................... 147
Netherlands Proteins@Work ............................................................................................ 149
ProteomeBinders initiative............................................................................................... 149
Rutgers Universitys Center for Integrative Proteomics Research .......................................... 150
Vanderbilt University's Center for Proteomics and Drug Actions............................................ 150

6. Pharmaceutical Applications of Proteomics ........................................ 151

Introduction..................................................................................................................... 151
Current drug discovery process and its limitations .......................................................... 151
Role of omics in drug discovery ....................................................................................... 152
Genomics-based drug discovery ....................................................................................... 152
Metabolomics technologies for drug discovery.................................................................... 153
Role of metabonomics in drug discovery ........................................................................... 153
Basis of proteomics approach to drug discovery ................................................................. 154
Proteins and drug action ............................................................................................ 154
Transcription-aided drug design .................................................................................. 155
Role of proteomic technologies in drug discovery ............................................................... 155
Liquid chromatography-based drug discovery ............................................................... 156
Capture compound mass spectrometry ........................................................................ 157
Protein-expression mapping by 2DGE .......................................................................... 157
Role of MALDI mass spectrometry in drug discovery ...................................................... 157
Tissue imaging mass spectrometry .............................................................................. 157
Companies using MALDI for drug discovery .................................................................. 159
Oxford Genome Anatomy Project ..................................................................................... 159
Proteins as drug targets .................................................................................................. 160
Ligands to capture the purine binding proteome ................................................................ 160
Chemical probes to interrogate key protein families for drug discovery ................................. 160
Global proteomics for pharmacodynamics ......................................................................... 161
CellCarta proteomics platform ....................................................................................... 161
ZeptoMARK protein profiling system............................................................................... 162
Role of proteomics in targeting disease pathways ........................................................... 162
Dynamic proteomics ....................................................................................................... 163
Identification of protein kinases as drug targets ................................................................. 163
Mechanisms of action of kinase inhibitors ..................................................................... 163
G-protein coupled receptors as drug targets ...................................................................... 164
Methods of study of GPCRs ......................................................................................... 164
Cell-based assays for GPCR ........................................................................................ 165
Companies involved in GPCR-based drug discovery ....................................................... 165
GPCR localization database......................................................................................... 166
Matrix metalloproteases as drug targets ........................................................................... 167
PDZ proteins as drug targets ........................................................................................... 167
Proteasome as drug target .............................................................................................. 168
Serine hydrolases as drug targets .................................................................................... 168
Targeting mTOR signaling pathway .................................................................................. 169
Targeting caspase-8 for anticancer therapeutics ................................................................ 170
Bioinformatic analysis of proteomics data for drug discovery .......................................... 170
Drug design based on structural proteomics .................................................................... 171
Protein crystallography for determining 3D structure of proteins .......................................... 171
Automated 3D protein modeling....................................................................................... 171
Drug targeting of flexible dynamic proteins .................................................................. 171
Companies involved in structure-based drug-design ........................................................... 172
Integration of genomics and proteomics for drug discovery ............................................ 173
Ligand-receptor binding ................................................................................................... 174
Role of proteomics in study of ligand-receptor binding ........................................................ 174
Aptamer protein binding ................................................................................................. 174
Systematic Evolution of Ligands by Exponential Enrichment ........................................... 174
Aptamers and high-throughput screening ..................................................................... 175
Nucleic Acid Biotools .................................................................................................. 175
Aptamer beacons ...................................................................................................... 176
Peptide aptamers ...................................................................................................... 176
Riboreporters for drug discovery ..................................................................................... 176
Target identification and validation ................................................................................. 177
Application of mass spectrometry for target identification ................................................... 177

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Gene knockout and gene suppression for validating protein targets ...................................... 178
Laser-mediated protein knockout for target validation ........................................................ 178
Integrated proteomics for drug discovery........................................................................ 178
High-throughput proteomics ............................................................................................ 179
Companies involved in high-throughput proteomics ............................................................ 179
Drug discovery through protein-protein interaction studies ............................................ 180
Protein-protein interaction as basis for drug target identification .......................................... 180
Protein-PCNA interaction as basis for drug design .............................................................. 181
Two-hybrid protein interaction technology for target identification........................................ 181
Biosensors for detection of small molecule-protein interactions ............................................ 182
Protein-protein interaction maps ...................................................................................... 183
ProNet (Myriad Genetics) ........................................................................................... 183
Hybrigenics' maps of protein-protein interactions .......................................................... 183
CellZome's functional map of protein-protein interactions .............................................. 183
Mapping of protein-protein interactions by mass spectrometry............................................. 184
Protein interaction map of Drosophila melanogaster ........................................................... 184
Protein-interaction map of Wellcome Trust Sanger Institute ................................................ 185
Protein-protein interactions as targets for therapeutic intervention ....................................... 185
Inhibition of protein-protein interactions by peptide aptamers ............................................. 185
Selective disruption of proteins by small molecules ............................................................ 186
Post-genomic combinatorial biology approach................................................................. 186
Differential proteomics .................................................................................................... 186
Shotgun proteomics ......................................................................................................... 187
Chemogenomics/chemoproteomics for drug discovery .................................................... 188
Chemoproteomics-based drug discovery ...................................................................... 189
Companies involved in chemogenomics/chemoproteomics .............................................. 190
Activity-based proteomics .......................................................................................... 191
Locus Discovery technology ........................................................................................ 191
Automated ligand identification system ........................................................................ 192
Expression proteomics: protein level quantification ........................................................ 192
Role of phage antibody libraries in target discovery ........................................................ 193
Analysis of posttranslational modification of proteins by MS ........................................... 193
Phosphoproteomics for drug discovery ............................................................................ 194
Application of glycoproteomics for drug discovery........................................................... 194
Role of carbohydrates in proteomics ................................................................................. 194
Challenges of glycoproteomics ......................................................................................... 195
Companies involved in glycoproteomics ............................................................................ 195
Role of protein microarrays/ biochips for drug discovery ................................................ 196
Protein microarrays vs DNA microarrays for high-throughput screening ................................ 196
BIA-MS biochip for protein-protein interactions .................................................................. 196
ProteinChip with Surface Enhanced Neat Desorption ........................................................... 197
Protein-domains microarrays ........................................................................................... 197
Some limitations of protein biochips ................................................................................. 197
Concluding remarks about role of proteomics in drug discovery ...................................... 198
RNA versus protein profiling as guide to drug development ............................................ 198
RNA as drug target......................................................................................................... 198
Combination of RNA and protein profiling .......................................................................... 199
RNA binding proteins ................................................................................................. 200
Toxicoproteomics ............................................................................................................. 200
Hepatotoxicity ............................................................................................................... 200
Nephrotoxicity ............................................................................................................... 201
Cardiotoxicity ................................................................................................................ 201
Neurotoxicity ................................................................................................................. 202
Protein/peptide therapeutics ........................................................................................... 202
Peptide-based drugs ....................................................................................................... 202
Phylomer peptides ........................................................................................................ 203
Cryptein-based therapeutics ............................................................................................ 203
Synthetic proteins and peptides as pharmaceuticals ........................................................... 204
Genetic immunization and proteomics ............................................................................. 204
Proteomics and gene therapy .......................................................................................... 205
Role of proteomics in clinical drug development .............................................................. 205
Pharmacoproteomics ...................................................................................................... 205
Role of proteomics in clinical drug safety........................................................................... 206

7. Application of Proteomics in Human Healthcare ................................. 207

Introduction..................................................................................................................... 207
Clinical proteomics........................................................................................................... 208
Definition and standards ................................................................................................. 208
Vermillion's Clinical Proteomics Program ........................................................................... 208
Pathophysiology of human diseases ................................................................................ 209
Diseases due to misfolding of proteins .............................................................................. 209

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Mechanism of protein folding ...................................................................................... 210

Nanoproteomics for study of misfolded proteins ............................................................ 211
Therapies for protein misfolding .................................................................................. 211
Intermediate filament proteins......................................................................................... 212
Significance of mitochondrial proteome in human disease ................................................... 213
Proteome of Saccharomyces cerevisiae mitochondria ..................................................... 213
Rat mitochondrial proteome ....................................................................................... 213
Proteomic approaches to biomarker identification ........................................................... 214
The ideal biomarker ....................................................................................................... 214
Proteomic technologies for biomarker discovery ................................................................. 214
MALDI mass spectrometry for biomarker discovery ....................................................... 215
BAMF Technology ................................................................................................... 215
Protein biochips/microarrays and biomarkers..................................................................... 216
Affinity proteomics for discovery of biomarkers .................................................................. 216
Antibody array-based biomarker discovery ................................................................... 216
Discovery of biomarkers by MAb microarray profiling ..................................................... 217
Tumor-specific serum peptidome patterns ......................................................................... 217
Search for protein biomarkers in body fluids ...................................................................... 218
Challenges and strategies for discovey of protein biomarkers in plasma ................................ 218
3-D structure of CD38 as a biomarker ......................................................................... 219
BD Free Flow Electrophoresis System ........................................................................ 219
Isotope tags for relative and absolute quantification ...................................................... 219
N-terminal peptide isolation from human plasma .......................................................... 220
Plasma protein microparticles as biomarkers ................................................................ 220
Proteome partitioning ................................................................................................ 221
SISCAPA method for quantitating proteins and peptides in plasma .................................. 221
Stable isotope tagging methods .................................................................................. 221
Technology to measure both the identity and size of the biomarker ................................. 222
Biomarkers in the urinary proteome ................................................................................. 222
Application of proteomics in molecular diagnosis ............................................................ 223
Proximity ligation assay .................................................................................................. 224
Protein patterns ............................................................................................................. 224
Proteomic tests on body fluids ......................................................................................... 224
Cyclical amplification of proteins ...................................................................................... 226
Applications of proteomics in infections .......................................................................... 226
MALDI-TOF MS for microbial identification ......................................................................... 226
Role of proteomics in virology .......................................................................................... 227
Study of interaction of proteins with viruses ................................................................. 227
Role of proteomics in bacteriology .................................................................................... 228
Epidemiology of bacterial infections ............................................................................. 228
Proteomic approach to bacterial pathogenesis............................................................... 228
Vaccines for bacterial infections .................................................................................. 228
Protein profiles associated with bacterial drug resistance................................................ 229
Analyses of the parasite proteome ................................................................................... 229
Application of proteomics in cystic fibrosis ...................................................................... 230
Proteomics of cardiovascular diseases ............................................................................. 230
Pathomechanism of cardiovascular diseases ...................................................................... 230
Study of cardiac mitochondrial proteome in myocardial ischemia ......................................... 231
Cardiac protein databases ............................................................................................... 231
Proteomics of dilated cardiomyopathy and heart failure ...................................................... 231
Proteomic biomarkers of cardiovascular diseases ............................................................... 232
Role of proteomics in cardioprotection .............................................................................. 232
Role of proteomics in heart transplantation ....................................................................... 232
Future of application of proteomics in cardiology ................................................................ 233
Proteomic technologies for research in pulmonary disorders .......................................... 233
Application of proteomics in renal disorders .................................................................... 234
Diagnosis of renal disorders ............................................................................................ 234
Proteomic biomarkers of acute kidney injury ..................................................................... 235
Cystatin C as biomarker of glomerular filtration rate ........................................................... 235
Protein biomarkers of nephritis ........................................................................................ 235
Proteomics and kidney stones .......................................................................................... 236
Proteomics of eye disorders ............................................................................................. 236
Proteomics of cataract .................................................................................................... 236
Proteomics of diabetic retinopathy ................................................................................... 237
Retinal dystrophies......................................................................................................... 237
Use of proteomics in inner ear disorders ......................................................................... 238
Use of proteomics in aging research ................................................................................ 238
Removal of altered cellular proteins in aging ..................................................................... 239
Alteration of glycoproteins during aging ............................................................................ 239
Proteomics and nutrition ................................................................................................. 239

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8. Oncoproteomics .................................................................................. 241

Introduction..................................................................................................................... 241
Proteomic technologies for study of cancer ..................................................................... 242
Application of CellCarta technology for oncology ................................................................ 242
Accentuation of differentially expressed proteins using phage technology .............................. 242
Identification of oncogenic tyrosine kinases using phosphoproteomics .................................. 242
Single-cell protein expression analysis by microfluidic techniques......................................... 243
Dynamic cell proteomics in response to a drug ............................................................. 243
Desorption electrospray ionization for cancer diagnosis ....................................................... 243
Proteomic analysis of cancer cell mitochondria ................................................................... 244
Mass spectrometry for identification of oncogenic chimeric proteins ...................................... 244
Id proteins as targets for cancer therapy .......................................................................... 245
Proteomic study of p53 ................................................................................................... 245
Human Tumor Gene Index .............................................................................................. 245
Integration of cancer genomics and proteomics ................................................................. 246
Laser capture microdissection technology and cancer proteomics ......................................... 246
Cancer tissue proteomics ................................................................................................ 247
Use of proteomics in cancers of various organ systems ................................................... 247
Proteomics of brain tumors ............................................................................................. 247
Proteomics of breast cancer ............................................................................................ 248
Proteomics of colorectal cancer ........................................................................................ 250
Proteomics of esophageal cancer ..................................................................................... 250
Proteomics of hepatic cancer ........................................................................................... 250
Proteomics of leukemia ................................................................................................... 251
Proteomics of lung cancer ............................................................................................... 252
Proteomics of pancreatic cancer ....................................................................................... 252
Proteomics of prostate cancer .......................................................................................... 252
Diagnostic use of cancer biomarkers ............................................................................... 253
Proteomic technologies for tumor biomarkers .................................................................... 254
Nuclear matrix proteins (NMPs) ....................................................................................... 254
Antiannexins as tumor markers in lung cancer ................................................................... 255
NCIs Network of Clinical Proteomic Technology Centers ................................................. 255
Proteomics and tumor immunology ................................................................................. 256
Proteomics and study of tumor invasiveness ................................................................... 257
Anticancer drug discovery and development.................................................................... 257
Kinase-targeted drug discovery in oncology....................................................................... 257
Anticancer drug targeting: functional proteomics screen of proteases ................................... 258
Small molecule inhibitors of cancer-related proteins ........................................................... 258
Role of proteomics in studying drug resistance in cancer ..................................................... 259
Future prospects of oncoproteomics ................................................................................ 259
Companies involved in application of proteomics to oncology ......................................... 259

9. Neuroproteomics ................................................................................ 261

Introduction .................................................................................................................. 261
Proteomics of prion diseases ........................................................................................... 261
Transmissible spongiform encephalopathies ...................................................................... 262
Creutzfeld-Jakob disease ............................................................................................ 262
Bovine spongiform encephalopathy ............................................................................. 263
Variant Creutzfeldt-Jakob disease ............................................................................... 263
Protein misfolding and neurodegenerative disorders ....................................................... 263
Ion channel link for protein-misfolding disease .................................................................. 263
Detection of misfolded proteins ........................................................................................ 264
Neurodegenerative disorders with protein abnormalities ..................................................... 264
Alzheimer disease ..................................................................................................... 266
Common denominators of Alzheimer and prion diseases ................................................ 266
Parkinson disease ..................................................................................................... 267
Amyotrophic lateral sclerosis ...................................................................................... 267
Proteomics and glutamate repeat disorders ....................................................................... 268
Proteomics and Huntington's disease ........................................................................... 268
Proteomics and demyelinating diseases .......................................................................... 269
Proteomics of neurogenetic disorders .............................................................................. 269
Fabry disease ................................................................................................................ 269
GM1 gangliosidosis ......................................................................................................... 270
Quantitative proteomics and familial hemiplegic migraine ................................................... 270
Proteomics of spinal muscular atrophy .............................................................................. 271
Proteomics of CNS trauma ............................................................................................... 271
Proteomics of traumatic brain injury ................................................................................. 271
Chronic traumatic encephalopathy and ALS ....................................................................... 272
Proteomics of CNS aging .................................................................................................. 272
Protein aggregation as a bimarker of aging ....................................................................... 272
Neuroproteomics of psychiatric disorders ........................................................................ 273

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Neuroproteomic of cocaine addiction ................................................................................ 273

Neurodiagnostics based on proteomics ............................................................................ 274
Disease-specific proteins in the cerebrospinal fluid ............................................................. 274
Tau proteins ............................................................................................................. 275
CNS tissue proteomics .................................................................................................... 275
Diagnosis of CNS disorders by examination of proteins in urine............................................ 277
Diagnosis of CNS disorders by examination of proteins in the blood...................................... 277
Serum pNF-H as biomarker of CNS damage.................................................................. 278
Proteomics of BBB ........................................................................................................... 278
Future prospects of neuroproteomics in neurology .......................................................... 279
HUPOs Pilot Brain Proteome Project ................................................................................. 280

10.

Commercial Aspects of Proteomics ............................................. 281

11.

Future of Proteomics .................................................................. 299

Introduction..................................................................................................................... 281
Potential markets for proteomic technologies ................................................................. 281
Bioinformatics markets for proteomics .............................................................................. 282
Markets for protein separation technologies....................................................................... 282
Markets for 2D gel electrophoresis .............................................................................. 282
Market trends in protein separation technolgies ............................................................ 283
Protein purification markets............................................................................................. 283
Mass spectrometry markets............................................................................................. 283
Markets for MALDI for drug discovery ............................................................................... 284
Markets for nuclear magnetic resonance spectroscopy ........................................................ 284
Market for structure-based drug design ............................................................................ 284
Markets for protein biomarkers ........................................................................................ 285
Markets for cell-based protein assays ............................................................................... 285
Protein biochip markets .................................................................................................. 285
Western blot markets ..................................................................................................... 285
Geographical distribution of proteomics technologies markets.............................................. 286
Business and strategic considerations ............................................................................. 286
Cost of protein structure determination............................................................................. 286
Opinion surveys of the scientist consumers of proteomic technologies .................................. 286
Opinions on mass spectrometry .................................................................................. 286
Opinions on bioinformatics and proteomic databases ..................................................... 287
Systems for in vivo study of protein-protein interactions ................................................ 287
Perceptions of the value of protein biochip/microfluidic systems ...................................... 287
Small versus big companies ............................................................................................ 287
Expansion in proteomics according to area of application .................................................... 288
Growth trends in cell-based protein assay market .............................................................. 288
Challenges for development of cell-based protein assays................................................ 288
Future trends and prospects of cell-based protein assays ............................................... 289
Strategic collaborations .................................................................................................. 289
Analysis of proteomics collaborations according to types of companies............................. 289
Types of proteomic collaborations ............................................................................... 290
Proteomics collaborations according to application areas ................................................ 290
Analysis of proteomics collaborations: types of technologies ........................................... 291
Collaborations based on protein biochip technology ....................................................... 291
Concluding remarks about proteomic collaborations ...................................................... 292
Proteomic patents .......................................................................................................... 292
Market drivers in proteomics ........................................................................................... 293
Needs of the pharmaceutical industry ............................................................................... 293
Need for outsourcing proteomic technologies ..................................................................... 293
Funding of proteomic companies and research ................................................................... 293
Technical advances in proteomics .................................................................................... 294
Changing trends in healthcare in future ............................................................................ 294
Challenges facing proteomics .......................................................................................... 294
Magnitude and complexity of the task ............................................................................... 294
Technical challenges ....................................................................................................... 295
Limitations of proteomics ................................................................................................ 295
Limitations of 2DGE ................................................................................................... 295
Limitations of mass spectrometry techniques ................................................................ 295
Complexity of the pharmaceutical proteomics ............................................................... 296
Unmet needs in proteomics ............................................................................................. 296

Genomics to proteomics................................................................................................... 299

Faster technologies ........................................................................................................ 299
FLEXGene repository ...................................................................................................... 299
Need for new proteomic technologies .............................................................................. 300
Emerging proteomic technologies .................................................................................... 301
Detection of alternative protein isoforms ........................................................................... 301

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Direct protein identification in large genomes by mass spectrometry .................................... 301

Proteome identification kits with stacked membranes ......................................................... 301
Vacuum deposition interface ............................................................................................ 302
In vitro protein biosynthesis ............................................................................................ 302
Proteome mining with adenosine triphosphate ................................................................... 302
Proteome-scale purification of human proteins from bacteria ............................................... 302
Proteostasis network ...................................................................................................... 303
Cytoproteomics.............................................................................................................. 303
Subcellular proteomics ............................................................................................... 303
Individual cell proteomics ........................................................................................... 304
Live cell proteomics ................................................................................................... 304
Fluorescent proteins for live-cell imaging ..................................................................... 305
Membrane proteomics .................................................................................................... 305
Identification of membrane proteins by tandem MS of protein ions .................................. 305
Solid state NMR for study of nanocrystalline membrane proteins ..................................... 306
Multiplex proteomics ...................................................................................................... 306
High-throughput for proteomics ....................................................................................... 306
Future directions for protein biochip application ................................................................. 307
Bioinformatics for proteomics .......................................................................................... 307
High-Throughput Crystallography Consortium .................................................................... 307
Study of protein folding by IBMs Blue Gene ...................................................................... 308
Study of proteins by atomic force microscopy .................................................................... 308
Population proteomics ..................................................................................................... 308
Comparative proteome analysis ....................................................................................... 309
Human Proteome Organization ........................................................................................ 309
Human Salivary Proteome................................................................................................ 310
Academic-commercial collaborations in proteomics......................................................... 310
Indiana Centers for Applied Protein Sciences ..................................................................... 310
Role of proteomics in the healthcare of the future ........................................................... 311
Proteomics and molecular medicine .................................................................................. 311
Proteodiagnostics ........................................................................................................... 311
Proteomics and personalized medicine .............................................................................. 312
Targeting the ubiquitin pathway for personalized therapy of cancer ................................. 312
Protein patterns and personalized medicine .................................................................. 312
Personalizing interferon therapy of hepatitis C virus ...................................................... 314
Protein biochips and personalized medicine .................................................................. 314
Combination of diagnostics and therapeutics ................................................................ 315
Future prospects ....................................................................................................... 315

12.

References .................................................................................. 317

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Landmarks in the evolution of proteomics ..................................................................... 19

Comparison of DNA and protein ................................................................................... 28
Comparison of mRNA and protein ................................................................................. 28
Methods of analysis at various levels of functional genomics ............................................ 34
Proteomics technologies .............................................................................................. 37
Protein separation technologies of selected companies .................................................... 42
Companies supplying mass spectrometry instruments..................................................... 45
Companies involved in cell-based protein assays ............................................................ 67
Methods used for the study of protein-protein interactions .............................................. 69
A selection of companies involved in protein-protein interaction studies ............................ 75
Companies involved in Western blotting ........................................................................ 90
Proteomic technologies used with laser capture microdissection ....................................... 92
Applications of protein biochip technology ....................................................................... 95
Selected companies involved in protein biochip/microarray technology ........................... 111
Proteomic databases and other Internet sources of proteomics information ..................... 119
Protein interaction databases available on the Internet ................................................. 124
Bioinformatic tools for proteomics from academic sources ............................................. 130
Selected companies involved in bioinformatics for proteomics ........................................ 131
Applications of proteomics in basic biological research .................................................. 132
A sampling of proteomics research projects in academic institutions ............................... 147
Pharmaceutical applications of proteomics ................................................................... 151
Selected companies relevant to MALDI-MS for drug discovery ........................................ 159
Selected companies involved in GPCR-based drug discovery .......................................... 166
Companies involved in drug design based on structural proteomics ................................ 172
Proteomic companies with high-throughput protein expression technologies .................... 179
Selected companies involved in chemogenomics/chemoproteomics ................................ 190
Companies involved in glycoproteomic technologies...................................................... 195

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7-1: Applications of proteomics in human healthcare ........................................................... 207

7-2: Eye disorders and proteomic approaches ..................................................................... 236
8-1: Companies involved in applications of proteomics to oncology ....................................... 260
9-1: Neurodegenerative diseases with underlying protein abnormality ................................... 264
9-2: Disease-specific proteins in the cerebrospinal fluid of patients........................................ 274
10-1: Potential markets for proteomic technologies 2011-2021 ............................................. 281
10-2: 2011 revenues of major companies from protein separation technologies ...................... 282
10-3: Geographical distribution of markets for proteomic technologies 2011-2021 .................. 286
11-1: Role of proteomics in personalizing strategies for cancer therapy .................................. 312

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1-1: A schematic miRNA pathway ...................................................................................... 22

1-2: Relationship of DNA, RNA and protein in the cell ............................................................. 29
1-3: Protein production pathway from gene expression to functional protein with controls. ........ 31
1-4: Parallels between functional genomics and proteomics ................................................... 32
2-1: Proteomics: flow from sample preparation to characterization......................................... 38
2-2: The central role of spectrometry in proteomics ............................................................. 45
2-3: Electrospray ionization (ESI) ...................................................................................... 46
2-4: Matrix-Assisted Laser Desorption/Ionization (MALDI) ..................................................... 47
2-5: Scheme of bio-bar-code assay .................................................................................... 62
2-6: A diagrammatic presentation of yeast two-hybrid system ............................................... 70
3-1: ProteinChip System ................................................................................................... 97
3-2: Surface plasma resonance (SPR) ............................................................................... 108
4-1: Role of bioinformatics in integrating genomic/proteomic-based drug discovery ............... 118
4-2: Bottom-up and top-down approaches for protein sequencing ........................................ 128
6-1: Drug discovery process ............................................................................................ 152
6-2: Regulatory changes induced by drugs and implemented at the proteins level. ................. 155
6-3: Relation of proteome to genome, diseases and drugs .................................................. 156
6-4: The mTOR pathways................................................................................................ 169
6-5: Steps in shotgun proteomics..................................................................................... 188
6-6: Chemogenomic approach to drug discovery (3-Dimensional Pharmaceuticals) ................ 189
8-1: Relation of oncoproteomics to other technologies ........................................................ 241
9-1: A scheme of proteomics applications in CNS drug discovery and development ................ 280
10-1: Types of companies involved in proteomics collaborations .......................................... 290
10-2: Types of collaborations: R & D, licensing or marketing ............................................... 290
10-3: Proteomics collaborations according to application areas ............................................ 291
10-4: Proteomics collaborations according to technologies .................................................. 291
10-5: Unmet needs in proteomics .................................................................................... 297
11-1: A scheme of the role of proteomics in personalized management of cancer ................... 314

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