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Proteomics in Practice# r3 S; x" v9 s: _4 Y8 l* e
A Guide to Successful Experimental Design

Abbreviations, Symbols, Units XV& B3 H& ]. E" X/ P! `; Y1 Q
Introduction 1
1 History 1
2 Critical Points 8
2.1 Challenges of the Protein Samples 8, C4 x+ u5 v  a2 T
2.1 Challenges of the Analysis Systems 11
3 Proteomics Strategies 12+ P( m# n; s7 U, Z2 {/ e
3.1 Proteome Mapping 12
3.2 Differential Analysis 12, o9 y* U7 o, b) T4 U
3.3 Time Point Experiments 13
3.4 Verification of Targets or Biomarkers 13
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  B2 W- z8 q) L, B- ^
4.2 Pooling of Samples: Yes or No? 14( Z4 [9 B# `0 {6 |* R3 q
4.3 Pre-fractionation of Samples: Yes or No? 142 ]% k5 m$ y  O* s' G: D
4.4 Which is the Best Workflow to Start With? 15
Part I: Proteomics Technology+ R: ?4 [3 ]1 O
1 Electrophoretic Techniques 19
1.1 The Principle of Electrophoresis and Some Methodological8 Z4 T& G" ]  ~3 y
Background 193 ~3 y# h1 {+ u6 W
1.1.1 Free Flow Electrophoretic Methods 20- c$ d& c8 p- A; V# G+ G2 H
1.1.2 Gels for Electrophoretic Techniques 21% e( a* i- F4 p- F) M: ~
1.1.3 Electroendosmosis Effects 21- P( ?% L4 W, k
1.2 Polyacrylamide Gel Electrophoresis 22

1.2.1 The Polyacrylamide Gel 22
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27+ S6 A5 [3 Y1 l# @7 Z* y
1.2.3 Blue Native Electrophoresis 32# Q/ I2 r# G; g1 F
1.2.4 Cationic Detergent Electrophoresis 34
1.3 Blotting 35
1.3.1 Electrophoretic Transfer 361 k; m+ i5 ^2 Q! O
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38( B: ~. r- @& w
1.4.1 Theoretical Background 396 J8 l" m5 j5 Y/ v6 c! i0 J# I
1.4.2 Preparation of IEF Gels 44# \! n1 Q! X, q. [4 r/ o: }0 N
1.4.3 Isoelectric Focusing in Proteomics 45
1.5 Two-dimensional Electrophoresis 53& r% s- U. m+ B  Y0 }' n
1.5.1 Sample Preparation 53
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68+ g( h. V# M2 p
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% @9 r) r$ O8 D7 @3 J$ \/ x. {
1.6 Image Analysis 125
1.6.1 Image Acquisition 125
1.6.2 Image Analysis and Evaluation 1299 H/ T7 h  |( o) _6 P+ m
1.6.3 Use of 2-D Electrophoresis Data 137
1.7 Spot Handling 1379 F% U5 G8 S2 }6 R- y+ F
1.7.1 Spot Picking 139; ~5 f& K/ _! F+ d( T
1.7.2 Protein Cleavage 141, q5 Q) B6 A9 s
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151
2.1.1 Ion Exchange Chromatography 153
2.1.2 Reversed Phase Chromatography 1620 r! R- W0 w: [! {. B3 a2 Q( R
2.1.3 Affinity Chromatography 167
2.1.4 Gel Filtration 1724 T& ?" W! Z7 `% D
2.2 Strategic Approach and General Applicability 174
2.3 Liquid Chromatography Techniques and Applications in Proteome. m' i3 [! N$ f) Y; z+ c- Y
Analysis 176
2.3.1 Peptide Separation 176. Y) B* ]$ h3 A, j  N5 U+ b
2.3.2 2DLC Peptide Separation 179
2.3.3 Affinity Chromatography and LC-MS/MS 1874 G! C% S* |4 S* i) @/ o/ P# R+ A
2.3.4 Protein Pre-fractionation 189: A  `  I, h. x4 `3 y6 H6 J+ b' L' ~
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 1948 Q' w: m1 r( W3 H+ a) k1 |
2.4.1 Sample Extraction and Preparation 196
2.4.2 Experimental Setup 197
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 1982 P( V# [( o0 K1 o
Contents
2.4.4 Reversed Phase Chromatography and
Protein Pre-fractionation 205
2.4.5 Fraction Size and Number of Fractions 2100 r# N% N6 I9 Z( |1 [. Y" |
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 215. @* c1 ?- ^3 e
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 218
3.1.2 Electrospray Ionization 222
3.2 Ion Separation 225( |2 b( E- C# H5 d
3.2.1 Time-of-Flight Analyzer 225. d6 {- ?4 f. P3 s7 o' z/ j/ y
3.2.2 Triple Quadrupole Analyzer 227
3.2.3 Quadrupole Ion Trap 228
3.2.4 Quadrupole Time-of-Flight 230
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 2312 U/ d* b8 p( E  T
3.2.6 TOF/TOF Analyzer 231) |2 A8 e8 v6 ]# ^2 F2 V
3.2.7 Fourier Transform Ion Cyclotron 232, R# y6 k6 Q# }
3.2.8 Orbitrap 233' r% [. z9 a% [8 z3 ^
3.3 Generating MS Data for Protein Identification 233
3.3.1 Peptide Mass Fingerprint 2343 S2 t+ ?9 R5 ^4 k5 z4 E& O
3.3.2 Peptide Mass Fingerprint Combined With Composition1 ?8 z( {: u" s' n9 J* n
Information 237
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
Information 238
3.3.4 Tandem Mass Spectrometry 242" }3 X# q; P. K7 U+ i! L
3.4 Protein Characterization 2583 b- c5 a2 p3 \0 I+ Q) n8 t. l
3.4.1 Phosphorylation Analysis 259/ P2 x6 y7 l) _4 a
3.4.2 Affinity Chromatography 260
3.4.3 Chemical Derivatization 2618 k2 u; Z/ p' ]
3.4.4 Glycosylation 263
3.5 Protein Quantification Using Mass Spectrometry 264
3.5.1 Stable Isotope Labeling Approaches 264" Z& ]" u. l3 B' _
3.5.2 Isotope-coded Affinity Tags 265' R/ R. i* I) O/ P- B+ ^1 T. M# C9 z
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 267* o, S/ k; [! X
3.5.5 iTRAQ 267
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 271. G. u/ p) i2 b9 q% i7 N& y% O
3.6.1 Bottom up Approach 2712 E1 k: ]/ Y- \' Z2 o9 g* o$ D3 l
3.6.2 Top down Approach 2727 B  F, G+ f7 M
4 Functional Proteomics: Studies of Protein–Protein Interactions 273
4.1 Non-immunological Methods 273: H% t& H# z1 y. }1 K, R9 ^
4.1.1 Separation of Intact Multi-protein Complexes 273# E1 G. B. }8 D
4.1.2 Probing with Interaction Partners 2732 G. G; I/ n8 B) T" c0 P/ p3 m  `
4.1.3 Surface Plasmon Resonance 274/ s7 t4 X+ u$ s! o! H
4.2 Antibody-based Techniques 275
4.2.1 Western Blotting and Dot Blots 275) _& h; z4 }3 @& N2 |* q
4.2.2 Protein Microarrays 276
Part II: Practical Manual of Proteome Analysis 279! w1 R2 k( M3 n& M+ S. {9 ]7 F
Equipment, Consumables, Reagents 281; M' n- t% N: P3 Q& k+ T5 E
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 2994 D1 _0 J& G1 Q. k1 ~
Step 3: Isoelectric Focusing 309' O4 |! H9 W, [) o4 K$ M
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357% s4 r! {5 p- `
Step 6: Staining of Gels 361
Step 7: Image Analysis and Evaluation of DIGE Gels 373$ J. ]" o! f  j3 Q& `4 E$ O- }
Step 8: Spot Excision 3839 y' d" o2 j/ `
Step 9: Sample Destaining 387  W3 }/ V/ @+ Q. A
Step 10: Protein Digestion 389/ v5 E1 k5 S( K$ r" i1 @- n) K: X( y
Step 11: Microscale Desalting and Concentrating of Sample 393
Step 12: Chemical Derivatization of the Peptide Digest 3973 f+ x; E6 T: |+ o7 \0 w
Step 13: MS Analysis 3998 Z, a  R( `- v/ P1 Z
Step 14: Calibration of MALDI-ToF MS 403
Step 15: Preparing for a Database Search 407
Part III: Trouble Shooting 411
1 Two-dimensional Electrophoresis 413
1.1 Sample Preparation 413
1.2 Isoelectric focusing in IGPG strips 4143 C9 L, h7 i( i6 [
1.3 SDS PAGE 4163 m+ s7 R4 v/ _
1.4 Staining 4177 v2 Y1 l5 y1 W: C8 H
1.5 DIGE Fluorescence Labeling 418( K5 b: ^( Q+ W8 ~, X8 a
1.6 Results in 2-D Electrophoresis 421
2 Mass Spectrometry 429
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