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Proteomics in Practice  t# C- p  k, k3 k8 a% m( R- k
A Guide to Successful Experimental Design8 Y4 c* y$ [1 p0 v" B( {4 a1 B

Abbreviations, Symbols, Units XV4 P+ b& J" X# U% D3 `" m& C
Introduction 1
1 History 1
2 Critical Points 8  J& b% S$ Y/ `
2.1 Challenges of the Protein Samples 8
2.1 Challenges of the Analysis Systems 11! t. u2 g$ X' W8 B  X9 [
3 Proteomics Strategies 12
3.1 Proteome Mapping 12
3.2 Differential Analysis 12% I) u7 a& Z% K
3.3 Time Point Experiments 130 V; N0 h6 H$ y. `* ?4 \
3.4 Verification of Targets or Biomarkers 13  L" D4 o7 n0 ]' W+ W% p# e* r
3.5 Integration of Results into Biological Context 13
3.6 Systems Biology 13
4 Concept of Experimental Planning 14
4.1 Biological Replicates 14% [4 b3 a7 D9 C$ g3 T9 \
4.2 Pooling of Samples: Yes or No? 146 }1 v, M% _, p3 F, z1 B! B" e
4.3 Pre-fractionation of Samples: Yes or No? 14" U8 e4 Z- U1 D
4.4 Which is the Best Workflow to Start With? 15
Part I: Proteomics Technology
1 Electrophoretic Techniques 19( q+ F% C# ^& T- ~' @$ D4 B
1.1 The Principle of Electrophoresis and Some Methodological
Background 193 O: f# [8 u6 r7 F$ e% w% u2 O0 ?' ]9 t
1.1.1 Free Flow Electrophoretic Methods 20# y; X/ I4 T! b
1.1.2 Gels for Electrophoretic Techniques 21
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 229 \8 \/ K9 z( K5 E, p
& o% W0 M/ R0 j3 n3 @
1.2.1 The Polyacrylamide Gel 22' M* I- \) b' S4 Q7 G! |1 S
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27
1.2.3 Blue Native Electrophoresis 32
1.2.4 Cationic Detergent Electrophoresis 34% @, P2 `/ C1 {/ `7 q- l
1.3 Blotting 35* G" R6 _+ d4 K
1.3.1 Electrophoretic Transfer 36) m. K2 h+ L2 [9 T8 w7 P! J
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38& |- q7 E7 R) u3 E/ G) v6 \
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 44% p0 D+ v6 p: R# S
1.4.3 Isoelectric Focusing in Proteomics 45
1.5 Two-dimensional Electrophoresis 53
1.5.1 Sample Preparation 53
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77
1.5.4 Second Dimension: SDS Electrophoresis 100- x) O# V% e# R) m6 k8 n! J$ m4 b
1.5.5 Detection of Protein Spots 119$ x$ |( @% U; Z: @# g! C
1.6 Image Analysis 125
1.6.1 Image Acquisition 1257 D% z8 g2 [. \
1.6.2 Image Analysis and Evaluation 129; L9 M1 l( y! N9 b) B6 A
1.6.3 Use of 2-D Electrophoresis Data 137+ f5 j. j* z7 S9 @+ Y: S
1.7 Spot Handling 1370 Z! I2 o5 T% _8 F
1.7.1 Spot Picking 1392 @% w( o! E: Y# f
1.7.2 Protein Cleavage 1417 ]3 |9 |3 _3 @$ l/ t8 j; \& v
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography
Techniques 1519 ?( y/ }4 A2 j6 X0 ]3 n
2.1.1 Ion Exchange Chromatography 153
2.1.2 Reversed Phase Chromatography 162
2.1.3 Affinity Chromatography 167' S; B- F( O8 c& ?
2.1.4 Gel Filtration 1723 a- Z# X1 z4 s' P
2.2 Strategic Approach and General Applicability 174
2.3 Liquid Chromatography Techniques and Applications in Proteome
Analysis 1763 C& H* H2 {7 p
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 179+ a7 G1 M( \! E
2.3.3 Affinity Chromatography and LC-MS/MS 1871 m! T* w2 ^3 j1 Z/ [
2.3.4 Protein Pre-fractionation 1892 z2 D2 s& |! d6 {: j
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 194( G. y, R% ^& L% Z5 `: ]. j" R
2.4.1 Sample Extraction and Preparation 196
2.4.2 Experimental Setup 197
2.4.3 Ion Exchange Chromatography and0 ~/ L5 h  n8 V  i) m$ F
Protein Pre-fractionation 198" E8 e4 _& P" z" p& i( _, j& g8 k( ~
Contents- R) G/ h0 [4 F! c  ]9 [
2.4.4 Reversed Phase Chromatography and" l8 C4 z( U* j0 f' A9 U
Protein Pre-fractionation 205
2.4.5 Fraction Size and Number of Fractions 210& L% ~  m4 i/ a
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 2156 b' p7 E$ e$ q8 {# Y
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 218
3.1.2 Electrospray Ionization 222
3.2 Ion Separation 225
3.2.1 Time-of-Flight Analyzer 2254 u7 e5 D- |# G7 ]; }5 H2 O
3.2.2 Triple Quadrupole Analyzer 227
3.2.3 Quadrupole Ion Trap 228
3.2.4 Quadrupole Time-of-Flight 230) v1 p+ k6 S. f1 i! {& T
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231' M4 b- {6 d, ~3 l& l0 E! V
3.2.6 TOF/TOF Analyzer 231
3.2.7 Fourier Transform Ion Cyclotron 232
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233" H; ], r" P) H! p
3.3.1 Peptide Mass Fingerprint 234
3.3.2 Peptide Mass Fingerprint Combined With Composition
Information 237' }9 p9 F: w5 }3 \* x& J" W6 ^6 y6 |
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence/ B4 c/ P& n3 T2 G3 u4 J% N
Information 238
3.3.4 Tandem Mass Spectrometry 242" q. Y0 S8 ~* W$ C) b0 X
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259
3.4.2 Affinity Chromatography 260- b% t3 C0 ]" t5 ?9 ?4 m
3.4.3 Chemical Derivatization 2612 K7 ]3 }+ w; S
3.4.4 Glycosylation 263
3.5 Protein Quantification Using Mass Spectrometry 264. ]) Y8 B% Y- B" s1 g4 O! @
3.5.1 Stable Isotope Labeling Approaches 264
3.5.2 Isotope-coded Affinity Tags 265
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 267
3.5.5 iTRAQ 267
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 271
3.6.1 Bottom up Approach 271
3.6.2 Top down Approach 272& }- S* d, m. E( U1 O8 R
4 Functional Proteomics: Studies of Protein–Protein Interactions 273% I  F" Y7 U% j( M# X/ Q9 ^% L) ~
4.1 Non-immunological Methods 273
4.1.1 Separation of Intact Multi-protein Complexes 273
4.1.2 Probing with Interaction Partners 2733 o" Q: l7 Z. {. }- U  v
4.1.3 Surface Plasmon Resonance 2743 V& |) \" \1 Y
4.2 Antibody-based Techniques 275) I( t6 p" ~; h( o0 B6 R/ d
4.2.1 Western Blotting and Dot Blots 275; d' C4 z; C# l7 j: Y
4.2.2 Protein Microarrays 276
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 2991 A; Y- b& I/ E, E9 I
Step 3: Isoelectric Focusing 309
Step 4: SDS Polyacrylamide Gel Electrophoresis 3238 K6 e, q5 _; x$ G/ Q8 {/ {
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
Step 6: Staining of Gels 361) }# ~  o( c0 C7 F
Step 7: Image Analysis and Evaluation of DIGE Gels 373
Step 8: Spot Excision 383
Step 9: Sample Destaining 387
Step 10: Protein Digestion 389
Step 11: Microscale Desalting and Concentrating of Sample 393
Step 12: Chemical Derivatization of the Peptide Digest 397
Step 13: MS Analysis 399
Step 14: Calibration of MALDI-ToF MS 403
Step 15: Preparing for a Database Search 4079 ~2 k) C! d( t! f2 o$ _
Part III: Trouble Shooting 4110 S2 r! j9 {" }. @. q- b; h
1 Two-dimensional Electrophoresis 413
1.1 Sample Preparation 4132 ~' Y! e! H8 H
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416
1.4 Staining 417, u7 h+ w; q- `& b0 ^
1.5 DIGE Fluorescence Labeling 418
1.6 Results in 2-D Electrophoresis 4218 G; v) H, w$ c% e
2 Mass Spectrometry 429( P' A* l) e0 m/ B* P

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