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Proteomics in Practice
A Guide to Successful Experimental Design
; D' @  i) ~! O( ^2 J$ C/ x+ ?
Abbreviations, Symbols, Units XV, |0 K3 c% J' L6 T9 Q
Introduction 1
1 History 1
2 Critical Points 8) S2 P) O9 e* V/ i# t
2.1 Challenges of the Protein Samples 89 g1 h$ {0 \- S% R% C) k6 T
2.1 Challenges of the Analysis Systems 115 L8 L4 C. N4 b1 s" p  J
3 Proteomics Strategies 125 w1 Q7 I* n' N9 F
3.1 Proteome Mapping 129 F& M& z" i  z) j. f2 I
3.2 Differential Analysis 122 x1 ^6 s8 C: x4 ^& _* X$ b
3.3 Time Point Experiments 13# U8 O2 q. c2 `: c9 b
3.4 Verification of Targets or Biomarkers 13$ a: S, D: @' a. P6 T5 P$ y
3.5 Integration of Results into Biological Context 13* ]- H) r* O! k1 v+ Q
3.6 Systems Biology 134 \# s! x& a( a& L& o
4 Concept of Experimental Planning 14/ Y0 f* E% H2 `) Y4 r* k8 w" t
4.1 Biological Replicates 14
4.2 Pooling of Samples: Yes or No? 14
4.3 Pre-fractionation of Samples: Yes or No? 14
4.4 Which is the Best Workflow to Start With? 159 y' E0 z* b) A+ G
Part I: Proteomics Technology  E6 X. e) D& e& v7 b; i8 [
1 Electrophoretic Techniques 199 A# O6 O! Y0 Z7 G" A; K
1.1 The Principle of Electrophoresis and Some Methodological! j3 m5 x+ Q) r2 `9 V( N
Background 19( x+ G) }: h0 c
1.1.1 Free Flow Electrophoretic Methods 20% x8 {8 h9 q* E0 z: _
1.1.2 Gels for Electrophoretic Techniques 21' D2 Y- E3 R7 Q" [* Z: l1 {
1.1.3 Electroendosmosis Effects 210 n0 R: d$ x6 _% Z! p6 B! E
1.2 Polyacrylamide Gel Electrophoresis 22
+ F0 K0 V7 c& M3 |
1.2.1 The Polyacrylamide Gel 22
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27, p- E4 F5 u9 L! e* L  S+ v' I
1.2.3 Blue Native Electrophoresis 32, e% y$ a$ g: N! j, {
1.2.4 Cationic Detergent Electrophoresis 347 H  F4 E. A5 a8 [8 Z0 ?
1.3 Blotting 353 _. C/ ?# _3 Z+ j
1.3.1 Electrophoretic Transfer 36
1.3.2 Protein Detection on the Membrane 364 g0 d1 Q! g( P9 \
1.4 Isoelectric Focusing 383 C; I3 K1 y8 ]3 J. w, `$ R
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 44
1.4.3 Isoelectric Focusing in Proteomics 45
1.5 Two-dimensional Electrophoresis 53+ Q' _) y! _! y$ ?
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
1.5.5 Detection of Protein Spots 119
1.6 Image Analysis 125( ^9 {( F% O% Y& u6 D
1.6.1 Image Acquisition 125/ m0 v& G7 b7 K' s7 F5 B
1.6.2 Image Analysis and Evaluation 129
1.6.3 Use of 2-D Electrophoresis Data 1378 Z0 s8 H/ s$ B6 @6 }- a0 q
1.7 Spot Handling 137
1.7.1 Spot Picking 139
1.7.2 Protein Cleavage 141, d# S5 O2 q) J
Liquid Chromatography Techniques 1513 j8 v- Y* ?+ }; e2 f
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151
2.1.1 Ion Exchange Chromatography 153
2.1.2 Reversed Phase Chromatography 162) m! I% [8 }$ o. C$ e, L
2.1.3 Affinity Chromatography 167
2.1.4 Gel Filtration 172/ i8 J, Q$ K7 t& t6 b
2.2 Strategic Approach and General Applicability 174
2.3 Liquid Chromatography Techniques and Applications in Proteome% n( `1 N9 k$ e$ d8 ^( q, ]: Q( G" ^' I
Analysis 176
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 179
2.3.3 Affinity Chromatography and LC-MS/MS 187
2.3.4 Protein Pre-fractionation 189
2.4 Practical Considerations and Application of LC-based Protein$ A* H4 M7 s2 I+ q, y
Pre-fractionation 194
2.4.1 Sample Extraction and Preparation 196$ K3 E+ u. I: u5 r
2.4.2 Experimental Setup 197# e9 D0 T9 N2 e* E
2.4.3 Ion Exchange Chromatography and: o: K7 H5 T% x6 W, l: E. F7 a
Protein Pre-fractionation 1988 {* f! e2 E8 j" K4 }9 b
Contents5 X5 |! X6 j& a0 @: E" a! {* v
2.4.4 Reversed Phase Chromatography and6 ]7 h6 ?5 u3 j5 `8 U7 {* u
Protein Pre-fractionation 205
2.4.5 Fraction Size and Number of Fractions 210$ V3 ?! H' s4 H' E
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 2155 @% n1 k- j- E1 `0 C' X
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 218! b" |( X' X+ `
3.1.2 Electrospray Ionization 222" A& E. s- `7 }9 }2 W
3.2 Ion Separation 225
3.2.1 Time-of-Flight Analyzer 225
3.2.2 Triple Quadrupole Analyzer 2278 o7 O( k( j8 `+ z
3.2.3 Quadrupole Ion Trap 228
3.2.4 Quadrupole Time-of-Flight 2301 ~& O# t  t" w' t6 Y; `' r) u
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231
3.2.6 TOF/TOF Analyzer 231* i5 }9 X# I" X* u0 s3 V# v. ]8 Q; h
3.2.7 Fourier Transform Ion Cyclotron 232
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233* s" C3 M( ]3 o! P' q
3.3.1 Peptide Mass Fingerprint 234# @! B! Z1 P" n7 J' e) f
3.3.2 Peptide Mass Fingerprint Combined With Composition$ _! b5 H! B( C# `8 @1 a8 f
Information 237
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence0 ]& m% a( l6 u( p$ M
Information 238* j3 x* V" [: K$ q* c- T; `
3.3.4 Tandem Mass Spectrometry 242
3.4 Protein Characterization 258' w0 _6 J9 K( s) `2 |3 i1 g
3.4.1 Phosphorylation Analysis 259: F+ I0 A4 W' p* I
3.4.2 Affinity Chromatography 260
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 263
3.5 Protein Quantification Using Mass Spectrometry 264; _9 Y: y: B7 m8 X1 S- i5 P/ K
3.5.1 Stable Isotope Labeling Approaches 264/ `. s1 y0 s$ G2 I$ p  r$ {
3.5.2 Isotope-coded Affinity Tags 265! p8 _7 r5 c: ^
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266( y4 }; ~$ s9 a! ^
3.5.4 AQUA 267
3.5.5 iTRAQ 267( q  V, M# p: Y: F3 c* v) B( u
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 271
3.6.1 Bottom up Approach 271" t' m' {0 Y2 h0 M3 }9 E
3.6.2 Top down Approach 2726 w0 ~- B5 \) b, ?0 B- I, R
4 Functional Proteomics: Studies of Protein–Protein Interactions 2730 l# W4 q6 Q! l- s. q: z
4.1 Non-immunological Methods 273
4.1.1 Separation of Intact Multi-protein Complexes 273
4.1.2 Probing with Interaction Partners 273* z7 z& W9 [# W3 V9 P
4.1.3 Surface Plasmon Resonance 274
4.2 Antibody-based Techniques 275
4.2.1 Western Blotting and Dot Blots 275
4.2.2 Protein Microarrays 276
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 281% |% G+ h+ P- Z+ D7 F) R  ~
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 299
Step 3: Isoelectric Focusing 309) G; l$ B) [  Q/ w
Step 4: SDS Polyacrylamide Gel Electrophoresis 323) G/ I! {  u. P
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
Step 6: Staining of Gels 361" Y0 b& a; C9 g- M; c3 b$ E- @
Step 7: Image Analysis and Evaluation of DIGE Gels 373* m) \/ c) v1 V5 a' ~
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) a: [* X" r  h2 A; E9 V5 i, m% a5 p
Step 13: MS Analysis 399
Step 14: Calibration of MALDI-ToF MS 403: w. z6 K& a; t# n5 m; t# f
Step 15: Preparing for a Database Search 407* B! h- R3 j1 r9 m& v+ h3 J6 |* x6 ^
Part III: Trouble Shooting 411
1 Two-dimensional Electrophoresis 413
1.1 Sample Preparation 413
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416( C5 a9 e) V' f6 b. ~; i
1.4 Staining 417- R* v1 J  K8 k7 V% v
1.5 DIGE Fluorescence Labeling 418$ c. q6 w* j$ o8 w# Q' ^* F& g, G0 K
1.6 Results in 2-D Electrophoresis 421
2 Mass Spectrometry 429/ Y: [$ ?* k) j
# \6 Y! W- K3 S
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