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Proteomics in Practice3 c/ T+ E, P! C2 Q% N8 X5 b% D5 s
A Guide to Successful Experimental Design

Abbreviations, Symbols, Units XV. A5 ^; @# c) u4 ^2 X
Introduction 1
1 History 1- R  \  E0 t+ W  c8 _+ k
2 Critical Points 8! T. v% \1 a) o* @- [
2.1 Challenges of the Protein Samples 8$ J* c" z9 D1 y' }6 z; U. H
2.1 Challenges of the Analysis Systems 11
3 Proteomics Strategies 12
3.1 Proteome Mapping 12
3.2 Differential Analysis 12
3.3 Time Point Experiments 136 k9 m- m+ k/ `7 t1 ~# x6 |
3.4 Verification of Targets or Biomarkers 13. w) F: O4 N) ?, N" o# ?
3.5 Integration of Results into Biological Context 13
3.6 Systems Biology 134 c' O# n- f9 w7 G; g0 j
4 Concept of Experimental Planning 14
4.1 Biological Replicates 14( ^/ A9 n+ q* @2 q" W" M
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? 15
Part I: Proteomics Technology
1 Electrophoretic Techniques 19( f& _; T2 m  K6 M& D
1.1 The Principle of Electrophoresis and Some Methodological  D# Z% G; z! t" m- x7 v/ _7 Q
Background 19
1.1.1 Free Flow Electrophoretic Methods 208 Y1 i7 [. x# `' e- z* `
1.1.2 Gels for Electrophoretic Techniques 21
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 22

1.2.1 The Polyacrylamide Gel 22- k2 q7 b& y8 E( T, r4 w* {
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27; l$ v( @' {* I& f/ v5 m
1.2.3 Blue Native Electrophoresis 32
1.2.4 Cationic Detergent Electrophoresis 34- e# X2 ^2 B: ]3 |2 O
1.3 Blotting 35& {, Z6 a, z4 A* Q6 W
1.3.1 Electrophoretic Transfer 369 |6 E. ]0 N* S0 N
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38* Z  x4 ]4 k1 T! u3 O7 R
1.4.1 Theoretical Background 39) V$ p9 V) _  J% {3 N
1.4.2 Preparation of IEF Gels 448 t% _3 Z& u8 D' V: X% j. |0 ]
1.4.3 Isoelectric Focusing in Proteomics 453 K& w$ G8 q$ c* h2 v* u
1.5 Two-dimensional Electrophoresis 536 l5 Y! m, v: k. ~; p
1.5.1 Sample Preparation 53$ Z0 F" }/ ]0 S5 U/ i! L4 z
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 681 D  |% N, X7 Y: T7 |5 _7 r/ m
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 1250 h. Z6 m' |! p$ ^# c" j
1.6.1 Image Acquisition 125
1.6.2 Image Analysis and Evaluation 129; f# Y; M& z; z! n4 F
1.6.3 Use of 2-D Electrophoresis Data 137
1.7 Spot Handling 137# m& z1 O+ t1 N# Y% E+ p9 P
1.7.1 Spot Picking 139
1.7.2 Protein Cleavage 141
Liquid Chromatography Techniques 1513 x# y9 E" M" t% Q2 l, f2 O1 N
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151
2.1.1 Ion Exchange Chromatography 1538 Y/ l' X9 H7 E7 ?& W2 O+ f9 h
2.1.2 Reversed Phase Chromatography 162! d; X, |5 i9 T' S7 M
2.1.3 Affinity Chromatography 167
2.1.4 Gel Filtration 1721 [# |7 G/ {8 J2 g3 G; s7 O7 n# ?2 V
2.2 Strategic Approach and General Applicability 174
2.3 Liquid Chromatography Techniques and Applications in Proteome
Analysis 176; g' X- p/ V  r1 o  K) g
2.3.1 Peptide Separation 1769 a; O9 x7 p6 E5 f- I* I- g* L
2.3.2 2DLC Peptide Separation 1799 r& C, g% y3 q5 ~5 w
2.3.3 Affinity Chromatography and LC-MS/MS 187
2.3.4 Protein Pre-fractionation 189' v1 {! x5 V8 u# n
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 194. c: ?5 z) {# Y
2.4.1 Sample Extraction and Preparation 196
2.4.2 Experimental Setup 197
2.4.3 Ion Exchange Chromatography and) m0 x8 ~; o: I( F# R! N
Protein Pre-fractionation 198* N' i) q2 k) w) c8 d
Contents
2.4.4 Reversed Phase Chromatography and( F' L: i5 ]0 g2 ?4 H, W8 H
Protein Pre-fractionation 205* e1 L4 i+ O; o& N( h1 C, _
2.4.5 Fraction Size and Number of Fractions 210
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 2154 M, A% _7 L1 ?" F( E
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 218& Q# F) w6 s1 a$ e
3.1.2 Electrospray Ionization 222
3.2 Ion Separation 225
3.2.1 Time-of-Flight Analyzer 225
3.2.2 Triple Quadrupole Analyzer 227" l' K" o1 |: y6 c( a: H( T
3.2.3 Quadrupole Ion Trap 228
3.2.4 Quadrupole Time-of-Flight 230
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231
3.2.6 TOF/TOF Analyzer 231) U0 `5 q8 G# J$ i8 C
3.2.7 Fourier Transform Ion Cyclotron 232
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233
3.3.1 Peptide Mass Fingerprint 234( x+ h  l. n/ [3 V4 k# ]0 @& c( o/ A
3.3.2 Peptide Mass Fingerprint Combined With Composition, ?7 H; a8 ~5 ]5 @1 J0 ^
Information 237  m9 r9 `: l) G% D& ]1 Z: Y8 K
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence* S5 q6 z. z: Y1 O
Information 238) M7 A4 i* S( ~% W: o) W0 O
3.3.4 Tandem Mass Spectrometry 242: o  r" o& Y+ s* l
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259, N- K+ \8 [/ L6 ]
3.4.2 Affinity Chromatography 260! i5 K, k9 z: _; v0 _
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 263
3.5 Protein Quantification Using Mass Spectrometry 264
3.5.1 Stable Isotope Labeling Approaches 2642 u: M9 W. H# h8 o& t' H9 Z  @
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# I8 m- f( F7 V4 i) e( L
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 271  D! k0 N6 N& i" ~1 |
3.6.1 Bottom up Approach 2715 ^! y/ z' p, t  v: |
3.6.2 Top down Approach 272
4 Functional Proteomics: Studies of Protein–Protein Interactions 273
4.1 Non-immunological Methods 273' n( Q- U$ }! _, N: f: G7 P- ~- m
4.1.1 Separation of Intact Multi-protein Complexes 273
4.1.2 Probing with Interaction Partners 273  n; f0 }; S% B
4.1.3 Surface Plasmon Resonance 274% j  h3 H9 P6 k7 `+ T4 ^9 y( U* M
4.2 Antibody-based Techniques 2757 t4 W7 B: s2 q* z' D- b; |; x4 F
4.2.1 Western Blotting and Dot Blots 275: J: ^5 X( A  `  U  ]. |
4.2.2 Protein Microarrays 276" @$ ]& ^3 A/ i9 U9 e
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 299, }( V# t6 u& J; F5 t% Y0 H* Y
Step 3: Isoelectric Focusing 309" s& W# p5 W$ V0 a# V- H8 |0 N; I
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357' Q% r/ z) o# X+ f; ?. u" D3 U
Step 6: Staining of Gels 3616 F6 L  J6 l% w) J
Step 7: Image Analysis and Evaluation of DIGE Gels 3739 U1 j7 q5 M1 R) A* b' F" F1 H
Step 8: Spot Excision 383
Step 9: Sample Destaining 387
Step 10: Protein Digestion 389' X$ j) S) ]' x4 o1 y
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 4076 F* A8 f9 c* O
Part III: Trouble Shooting 411+ [$ n8 }5 o6 u# P- _! K4 Z; D
1 Two-dimensional Electrophoresis 4133 K+ X5 l7 n# L% l+ W7 U$ I
1.1 Sample Preparation 413; Z% Q3 W5 K- a
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416
1.4 Staining 4172 B" D7 o0 }  L+ |. @3 u
1.5 DIGE Fluorescence Labeling 418
1.6 Results in 2-D Electrophoresis 421  j4 s$ U/ Y0 X# ]3 E
2 Mass Spectrometry 429
6 V: d/ p' A& \, p, K. g# {3 k3 j
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