r

Principles of Proteomics Second Edition

Richard M. Twyman

r0 Garland Science \...:),._) Taylor & Francis Group

NEW YORK AND LONDON

VIII -

Contents

Chapter 1 The origin and scope The four major limitations of 2DGE are

of proteomics 1 resolution, sensitivity, representation, and compatibility with automated protein analysis 30

1.1 INTRODUCTION 1 The resolution of 2DGE can be improved with

1.2 THE BIRTH OF LARGE-SCALE BIOLOGY giant gels, zoom gels, and modified gradients,

AND THE "OMICS" ERA 1 or by pre-fractionating the sample 30

1.3 THE GENOME, TRANSCRIPTOME, The sensitivity of 2DGE depends on the visualization

PROTEOME, AND METABOLOME 6 of minor protein spots, which can be masked by abundant proteins 31

1.4 FUNCTIONAL GENOMICS 8 The representation of hydrophobic proteins is Transcriptomics is the systematic, global an intractable problem reflecting the buffers analysis of mRNA 8 required for isoelectric focusing 32

Large-scale mutagenesis and interference can Downstream mass spectrometry requires spot also determine the functions of genes on a analysis and picking 34 global scale 11 2.5 PRINCIPLES OF MULTIDIMENSIONAL

1.5 THE NEED FOR PROTEOMICS 15 LIQUID CHROMATOGRAPHY 34

1.6 THE SCOPE OF PROTEOMICS 17 Protein and peptide separation by

Protein identification and quantitation are chromatography relies on differing affinity

the most fundamental aspects of proteomic for stationary and mobile phases 34

analysis 17 Affinity chromatography exploits the specific

Important functional data can be gained from binding characteristics of proteins and/or

sequence and structural analysis 18 peptides 36

Interaction proteomics and activity-based Size exclusion chromatography sieves

proteomics can help to link proteins into molecules on the basis of their size 36

functional networks 19 ion exchange chromatography exploits

1.7 CURRENT CHALLENGES IN PROTEOMICS 20 differences in net charge 37

Reversed-phase chromatography and hydrophobic interaction chromatography

Chapter 2 Strategies for exploit the affinity between peptides and hydrophobic resins 38

protein separation 23 2.6 MULTIDIMENSIONAL LIQUID

2.1 INTRODUCTION 23 CHROMATOGRAPHY STRATEGIES IN

2.2 GENERAL PRINCIPLES OF PROTEIN PROTEOMICS 39

SEPARATION IN PROTEOMICS 23 Multidimensional liquid chromatography is

2.3 PRINCIPLES OF TWO-DIMENSIONAL GEL more versatile and more easily automated than

ELECTROPHORESIS 25 2DGE but lacks a visual dimension 39

Electrophoresis separates proteins by mass The most useful MDLC systems achieve optimal peak capacity by exploiting orthogonal

and charge 25 separations that have internally compatible \soelectric focusing separates proteins by buffers 40 charge irrespective of mass 26 MudPIT shows how MDLC has evolved from a SDS-PAG E separates proteins by mass laborious technique to virtually hands-free irrespective of charge 28 operation 41

2.4 THE APPLICATION OF 2DGE IN RP-RPLC and HIL\C-RP systems offer advantages PROTEOMICS 29 for the separation of certain types of peptide

The four major advantages of 2DGE are mixtures 44

robustness, reproducibility, visualization, and Affinity chromatography is combined with MDLC compatibility with downstream microanalysis 29 to achieve the simplification of peptide mixtures 44

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" CONTENTS ix

Chapter 3 Strategies for 4.3 MULTIPLEXED IN-GEL PROTEOMICS 75 protein identification 47 Difference in-gel electrophoresis involves the

3.1 INTRODUCTION 47 simultaneous separation of comparative protein samples labeled with different

3.2 PROTEIN IDENTIFICATION WITH fluorophores 75 ANTIBODIES 47 Parallel analysis with multiple dyes can

3.3 DETERMINATION OF PROTEIN ·also be used to identify particular structural

SEQUENCES BY CHEMICAL or functional groups of proteins 76

DEGRADATION 48 4.4 QUANTITATIVE MASS SPECTROMETRY 77 Complete hydrolysis allows protein sequences Label-free quantitation may be based on

to be inferred from the content of the resulting spectral counting or the comparison of signal amino acid pool 48 intensities across samples in a narrow mlz

Edman degradation was the first general range 77

method for the de novo sequencing of Label-based quantitation involves the

proteins 49 incorporation of labels that allow

Edman degradation was the first protein corresponding peptides in different

identification method to be applied in samples to be identified by a specific change

proteomics, but it is difficult to apply on a in mass 77

large scale so ICAT reagents are used for the selective

3.4 MASS SPECTROMETRY-BASIC labeling of proteins or peptides 79

PRINCIPLES AND INSTRUMENTATION 52 Proteins and peptides can also be labeled nonselectively 80

Mass spectrometry is based on the separation Isobaric tagging allows protein quantitation

of molecules according to their mass/charge ratio 52 by the detection of reporter ions 80

The integration of mass spectrometry jnto Metabolic labeling introduces the label before

proteomics required the development of sample preparation but is limited to simple

soft ionization methods to prevent random organisms and cultured cells 83

fragmentation 52 Controlled fragmentation is used to break Chapter 5 The analysis of peptide bonds and generate fragment ions 53 Five principal types of mass analyzer are

protein sequences 87

commonly used in proteomics 54 5.1 INTRODUCTION 87 3.5 PROTEIN IDENTIFICATION USING DATA 5.2 PROTEIN FAMILIES AND

FROM MASS SPECTRA 58 EVOLUTIONARY RELATIONSHIPS 89 Peptide mass fingerprinting correlates e Evolutionary relationships between proteins xperimental and theoretical intact peptide are based on homology 89 masses 58 The function of a protein can often be Shotgun proteomics can be combined with predicted from its sequence 92 database searches based on uninterpreted 5.3 PRINCIPLES OF PROTEIN SEQUENCE spectra 61

COMPARISON 93 .. MS/MS spectra can be used to derive protein

Protein sequences can be compared in terms sequences de novo 61

of identity and similarity 93 Homologous sequences are found by pairwise

Chapter 4 Strategies for similarity searching 93

protein quantitation 69 Substitution score matrices rank the importance of different substitutions 96

4.1 INTRODUCTION 69 Sequence alignment scores depend on

4.2 QUANTITATIVE PROTEOMICS BASED sequence length 98

ON 2DGE 70 Multiple alignments provide more information

The quantitation of proteins in two-dimensional about key sequence elements 98

gels involves the creation of digital data from 5.4 STRATEGIES TO FIND MORE DISTANT analog images 70 RELATIONSHIPS 100 Spot detection, quantitation, and comparison PSI-BLAST uses sequence profiles to carry out can be challenging without human intervention 71 iterative searches 100

X CONTENTS

Pattern recognition methods incorporate Affinity-based biochemical methods provide conserved sequence signatures 101 direct evidence that proteins can interact 138

5.5 THE RISK OF FALSE-POSITIVE Interactions between proteins in vitro and

ANNOTATIONS 104 in vivo can be established by resonance energy transfer 142 Surface plasmon resonance can indicate the

Chapter 6 The analysis of mass of interacting proteins 142

protein structures 107 7.3 LIBRARY-BASED METHODS FOR THE GLOBAL ANALYSIS OF BINARY

6.1 INTRODUCTION 107 INTERACTIONS 143 6.2 STRUCTURAL GENOMICS AND 7.4 TWO-HYBRID/PROTEIN

STRUCTURE SPACE 110 COMPLEMENTATION ASSAYS 145 Coverage of structure space is currently The yeast two-hybrid system works by uneven 110 assembling a transcription factor from two Structure and function are not always related 113 inactive fusion proteins 145

6.3 TECHNIQUES FOR SOLVING PROTEIN Several large-scale interaction screens have

STRUCTURES 114 been carried out using different yeast

X-ray diffraction requires well-ordered protein two-hybrid screening strategies 146

crystals 114 Conventional yeast two-hybrid screens have

NMR spectroscopy exploits the magnetic a significant error rate 148

properties of certain atomic nuclei 116 7.5 MODIFIED TWO-HYBRID SYSTEMS

Additional methods for structural analysis FOR MEMBRANE, CYTOSOLIC, AND

mainly provide supporting data 118 EXTRACELLULAR PROTEINS 149

6.4 PROTEIN STRUCTURE PREDICTION 119 7.6 BACTERIAL AND MAMMALIAN

Structural predictions can bridge the gap TWO-HYBRID SYSTEMS 150

between sequence and structure 119 7.7 LUMIER AND MAPPIT HIGH-

Protein secondary structures can be THROUGHPUT TWO-HYBRID predicted from sequence data 120 PLATFORMS 151

Tertiary structures can be predicted by 7.8 ADAPTED HYBRID ASSAYS FOR comparative modeling if a template DIFFERENT TYPES OF INTERACTIONS 152 structure is available 122 7.9 SYSTEMATIC COMPLEX ANALYSIS Ab initio prediction methods attempt to BY TANDEM AFFINITY PURIFICATION-construct structures from first principles 123 MASS SPECTROMETRY 153 Fold recognition (threading) is based on 7.10 ANALYSIS OF PROTEIN INTERACTION similarities between nonhomologous folds 123

DATA 155 6.5 COMPARISON OF PROTEIN

7.11 PROTEIN INTERACTION MAPS STRUCTURES 124 156

6.6 STRUCTURAL CLASSIFICATION OF 7.12 PROTEIN INTERACTIONS WITH SMALL

PROTEINS 125 MOLECULES 158

6.7 GLOBAL STRUCTURAL GENOMICS INITIATIVES 126 Chapter 8 Protein

modification in proteomics 165

Chapte 7 Interaction 8.1 INTRODUCTION 165

proteomics 131 8.2 METHODS FOR THE DETECTION OF

7.1 INTRODUCTION 131 POST-TRANSLATIONAL MODIFICATIONS 167

7.2 METHODS TO STUDY 8.3 ENRICHMENT STRATEGIES FOR

PROTEIN-PROTEIN INTERACTIONS 134 MODIFIED PROTEINS AND PEPTIDES 168

Genetic methods suggest interactions from 8.4 PHOSPHOPROTEOMICS 170

the combined effects of two mutations in Protein phosphorylation is a key regulatory the same cell or organism 134 mechanism 170 Protein interactions can be suggested by Separated phosphoproteins can be detected comparative genomics and homology transfer 135 with specific staining reagents 172

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CONTENTS xi --Sample preparation for phosphoprotein Cell-free expression systems allow the direct analysis typically involves enrichment synthesis of protein arrays in situ 197 using antibodies or strongly cationic

173 9.5 THE MANUFACTURE OF FUNCTIONAL chromatography resins

PROTEIN MICROARRAYS-PROTEIN 8.5 ANALYSIS OF PHOSPHOPROTEINS BY IMMOBILIZATION 201

MASS SPECTROMETRY 176 9.6 THE DETECTION OF PROTEINS ON

A combination of Edman degradation and MICROARRAYS 203 mass spectrometry can be used to map

Methods that require labels can involve phosphorylation sites 176 either direct or indirect detection 203

Intact phosphopeptide ions can be identified 176 Label-free methods do not affect the by MALDI-TOF mass spectrometry

intrinsic properties of interacting proteins 204 Phosphopeptides yield diagnostic marker ions

177 9.7 EMERGING PROTEIN CHIP and neutral loss products TECHNOLOGIES 207

8.6 QUANTITATIVE ANALYSIS OF Bead and particle arrays in solution represent PHOSPHOPROTEINS 180 the next generation of protein microarrays 207

8.7 GLYCOPROTEOMICS 181 Cell and tissue arrays allow the direct Glycoproteins represent more than half of analysis of proteins in vivo 207 the eukaryotic proteome 181

Glycans play important roles in protein

Applications stability, activity, and localization, and are Chapter 10 important indicators of disease 183 of proteomics 211 Conventional glycoanalysis involves the use of enzymes that remove specific glycan 10.1 INTRODUCTION 211 groups and the separation of glycoproteins

184 10.2 DIAGNOSTIC APPLICATIONS OF

by electrophoresis PROTEOMICS 212 Glycoprotein-specific staining allows the

187 Proteomics is used to identify biomarkers of

glycoprotein to be studied by 2DGE disease states 212 There are two principal methods for Biomarkers can be discovered by finding glycoprotein enrichment that have

188 plus/minus or quantitative differences

complementary uses between samples 215 Mass spectrometry is used for the high- More sensitive techniques can be used to throughput identification and identify biomarker profiles 21 8 characterization of glycoproteins 189

10.3 APPLICATIONS OF PROTEOMICS IN DRUG DEVELOPMENT 219

Chapter 9 Protein microarrays 191 Proteomics can help to select drug targets and develop lead compounds 219

9.1 INTRODUCTION 191 Proteomics is also useful for target validation 222 9.2 THE EVOLUTION OF PROTEIN Chemical proteomics can be used to select

MICROARRAYS 191 and develop lead compounds 222

9.3 DIFFERENT TYPES OF PROTEIN Proteomics can be used to assess drug MICROARRAYS 193 toxicity during clinical development 224

Analytical, functional, and reverse microarrays 10.4 PROTEOMICS IN AGRICULTURE 225 are distinguished by their purpose and the Proteomics provides novel markers in plant nature of the interacting components 193 breeding and genetics 225 Analytical microarrays contain antibodies Proteomics can be used for the analysis of or other capture reagents 194 genetically modified plants 227 Functional protein microarrays can be used

10.5 PROTEOMICS IN INDUSTRY-to study a wide range of biochemical

196 IMPROVING THE YIELD OF functions SECONDARY METABOLISM 228

9.4 THE MANUFACTURE OF FUNCTIONAL PROTEIN MICROARRAYS-PROTEIN SYNTHESIS 197 Glossary 231 Proteins can be synthesized by the parallel construction of many expression vectors 197 Index 248