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本帖最后由细胞海洋于 2010-6-28 11:09 编辑 3 b/ i$ U+ t$ f1 V3 g
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Proteomics in Practice
A Guide to Successful Experimental Design7 |2 q. o8 V8 M$ J/ ?# G. D
1 }6 F0 M  m! ?5 F8 y0 \' T
Abbreviations, Symbols, Units XV! c3 x3 O1 f# U, D8 W/ F7 [. r4 J
Introduction 1( L/ D; D& G9 ~% X, m" y
1 History 10 F8 u  u3 w; H6 u
2 Critical Points 82 O5 j+ G' i) ]$ c5 V9 |$ B% y* d
2.1 Challenges of the Protein Samples 8# w4 q# x4 a8 I( G6 m% c1 ?
2.1 Challenges of the Analysis Systems 11
3 Proteomics Strategies 129 }4 H7 }" X# X' @$ T. w
3.1 Proteome Mapping 12
3.2 Differential Analysis 12
3.3 Time Point Experiments 13# P, H7 p" s% P( a' W( B
3.4 Verification of Targets or Biomarkers 13
3.5 Integration of Results into Biological Context 13& q9 \7 S/ r( q6 ~
3.6 Systems Biology 13
4 Concept of Experimental Planning 14
4.1 Biological Replicates 147 d8 L) i7 `( D
4.2 Pooling of Samples: Yes or No? 140 r% O- y7 q  s& [: l- n5 g4 F' K
4.3 Pre-fractionation of Samples: Yes or No? 14
4.4 Which is the Best Workflow to Start With? 15
Part I: Proteomics Technology3 M3 k. P! x; v' _
1 Electrophoretic Techniques 19
1.1 The Principle of Electrophoresis and Some Methodological& m8 w& t) U' o8 ]2 N
Background 19
1.1.1 Free Flow Electrophoretic Methods 20& T, O4 i$ j8 J/ X4 h
1.1.2 Gels for Electrophoretic Techniques 219 R# [2 h0 T( P: k. \
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 22

1.2.1 The Polyacrylamide Gel 22
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27) ?& L/ r! u% ~. s
1.2.3 Blue Native Electrophoresis 32
1.2.4 Cationic Detergent Electrophoresis 34
1.3 Blotting 35
1.3.1 Electrophoretic Transfer 36
1.3.2 Protein Detection on the Membrane 360 u, k3 D3 M$ ]( }; `
1.4 Isoelectric Focusing 380 g" s" z1 p9 v; f+ _; g( Y, C$ v
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 44  e. m# q/ o' M5 O* a
1.4.3 Isoelectric Focusing in Proteomics 45) W5 D2 x+ {$ Y+ N9 m
1.5 Two-dimensional Electrophoresis 53
1.5.1 Sample Preparation 536 R& ?* x: u9 D4 U7 v
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77  L8 |' W9 C/ N; z0 B; Z  m; a
1.5.4 Second Dimension: SDS Electrophoresis 100% {6 U5 i4 z2 \" h
1.5.5 Detection of Protein Spots 119
1.6 Image Analysis 125
1.6.1 Image Acquisition 1253 q% T' S& D; Q5 ~" l( y# K
1.6.2 Image Analysis and Evaluation 129$ W2 v9 L# A# C$ _
1.6.3 Use of 2-D Electrophoresis Data 137
1.7 Spot Handling 137' \, H) V; {/ ?4 J" N6 ^
1.7.1 Spot Picking 139
1.7.2 Protein Cleavage 141$ s, K) `7 \$ g! C# B
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151# `+ r7 a4 }, E3 q7 f
2.1.1 Ion Exchange Chromatography 153) D7 X. A; O9 W
2.1.2 Reversed Phase Chromatography 162
2.1.3 Affinity Chromatography 167
2.1.4 Gel Filtration 1727 A' t9 s- ^! S& e
2.2 Strategic Approach and General Applicability 174# j: F0 [$ H, y. G- n* ]
2.3 Liquid Chromatography Techniques and Applications in Proteome6 M" I& x5 {4 ~9 u3 c
Analysis 1762 ?, H' g4 L& [5 E7 g1 {
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 179
2.3.3 Affinity Chromatography and LC-MS/MS 187# C4 V$ C5 S2 _/ N
2.3.4 Protein Pre-fractionation 189
2.4 Practical Considerations and Application of LC-based Protein* h( a% p1 y' ~( X0 F/ {
Pre-fractionation 194
2.4.1 Sample Extraction and Preparation 196
2.4.2 Experimental Setup 197- v5 ]5 V; _) I& c8 j+ K: u! F+ k) G
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 198: E  I4 F* G, C$ a8 s8 w
Contents4 T  e( s; c5 }: \
2.4.4 Reversed Phase Chromatography and# @( E2 H7 k/ s$ K( ]/ J+ M: T
Protein Pre-fractionation 2052 D; k8 L0 Q7 E
2.4.5 Fraction Size and Number of Fractions 210
2.5 Critical Review and Outlook 2118 @7 A9 B0 `2 `2 r1 ]# P# D
3 Mass Spectrometry 215
3.1 Ionization 218# E$ E8 j' J3 W, h6 m' H0 `% }
3.1.1 Matrix Assisted Laser Desorption Ionization 218' N) y* m% O, d0 F  h
3.1.2 Electrospray Ionization 222  {8 j! f! P$ R* U, e
3.2 Ion Separation 2258 X. j; w; C- X# S# G1 a  d
3.2.1 Time-of-Flight Analyzer 225
3.2.2 Triple Quadrupole Analyzer 227
3.2.3 Quadrupole Ion Trap 228) T9 e1 q& p9 b" p
3.2.4 Quadrupole Time-of-Flight 230. i% f4 S' u! q: Y; t* F
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231
3.2.6 TOF/TOF Analyzer 2312 x$ k4 ?" k9 h% ^  P2 I8 B
3.2.7 Fourier Transform Ion Cyclotron 232
3.2.8 Orbitrap 233  b. u7 O* Q4 d. K) H7 @. H
3.3 Generating MS Data for Protein Identification 2337 {4 }* s# M+ X8 G# i6 u5 s
3.3.1 Peptide Mass Fingerprint 234
3.3.2 Peptide Mass Fingerprint Combined With Composition
Information 237
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence8 n( [8 ~* j7 o0 @4 N1 h
Information 238: S$ p6 E8 C( F- s1 D2 A
3.3.4 Tandem Mass Spectrometry 242
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259
3.4.2 Affinity Chromatography 260( p, b8 U2 j1 r8 V
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 264* B8 ~9 Y' p! L) z2 k" H3 e! G
3.5.2 Isotope-coded Affinity Tags 265* _, J. ]* q$ d
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 2677 R& j) E+ _+ v9 @  i+ P
3.5.5 iTRAQ 267
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 2718 j9 v. D: a( S: C
3.6.1 Bottom up Approach 271
3.6.2 Top down Approach 2728 O  S7 |# P: _7 w. F$ c; t( B) n" l
4 Functional Proteomics: Studies of Protein–Protein Interactions 273, c, M: M) _' M3 Z3 o
4.1 Non-immunological Methods 273/ I. U  E* _! j- r/ ]
4.1.1 Separation of Intact Multi-protein Complexes 273
4.1.2 Probing with Interaction Partners 2737 I  i# y6 L. F. T2 p8 [7 F9 G
4.1.3 Surface Plasmon Resonance 2744 F# C4 @2 V" W% i* d3 E$ r+ J
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 2795 r" [/ p, K2 L/ v8 K3 W+ C: N& F
Equipment, Consumables, Reagents 281$ T6 I* g$ ?7 Z6 p- @0 U2 o( k) E
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 299. l( `: c# v6 Q. L- N) E
Step 3: Isoelectric Focusing 3096 k! V- y: a: b, D5 v# a
Step 4: SDS Polyacrylamide Gel Electrophoresis 3236 q9 R, r. k3 Q! }
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357) B* C. \, w! i' `
Step 6: Staining of Gels 361# Q) h2 ~, S2 o, e3 o4 _
Step 7: Image Analysis and Evaluation of DIGE Gels 3738 G7 F4 _: c! S$ p& V
Step 8: Spot Excision 383; P5 n& A) L$ g: q  _
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 4034 ]) s2 a" W9 `6 r6 ]0 J/ J" z6 n$ h
Step 15: Preparing for a Database Search 407) I- v6 B( J9 v+ l
Part III: Trouble Shooting 411/ O4 d- e# C/ J. \! |- Y7 F' S
1 Two-dimensional Electrophoresis 413. C) n& v8 r1 ^- Z& D+ k0 X
1.1 Sample Preparation 413
1.2 Isoelectric focusing in IGPG strips 4142 i# ?0 b- o: {
1.3 SDS PAGE 416
1.4 Staining 417
1.5 DIGE Fluorescence Labeling 418( f: J) P' Q" P
1.6 Results in 2-D Electrophoresis 421$ ?! x6 e0 X' T. C, J
2 Mass Spectrometry 429, z( Y* l& G0 j& |" F" ^: }

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