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本帖最后由细胞海洋于 2010-6-28 11:09 编辑 5 Z8 b7 z1 h; \! s. y
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Proteomics in Practice, G$ H6 S6 y1 Q7 ?9 t! ^' i
A Guide to Successful Experimental Design) j2 {0 W6 X9 p7 s( j
/ x- G# H7 q# R$ O1 x8 ]8 I1 _1 Q8 o
Abbreviations, Symbols, Units XV
Introduction 1
1 History 1! X6 a! |- ?: x0 [4 @) ]
2 Critical Points 8
2.1 Challenges of the Protein Samples 8
2.1 Challenges of the Analysis Systems 11; M7 X5 O/ y9 e8 ~# ?8 l
3 Proteomics Strategies 12* |% _. _9 C: I' Q) T( E- E
3.1 Proteome Mapping 12
3.2 Differential Analysis 12
3.3 Time Point Experiments 13
3.4 Verification of Targets or Biomarkers 13: C0 h3 z+ E& S4 Y0 o) q2 r
3.5 Integration of Results into Biological Context 13) Z: W& _4 D6 k! M& o0 G) c& g
3.6 Systems Biology 13
4 Concept of Experimental Planning 141 w' g6 A5 S% y( `) `
4.1 Biological Replicates 14
4.2 Pooling of Samples: Yes or No? 14# p5 v7 l7 ]: X% `  f
4.3 Pre-fractionation of Samples: Yes or No? 14
4.4 Which is the Best Workflow to Start With? 15& o* L& {% v2 N8 z/ s; K
Part I: Proteomics Technology" i' n6 d: N, ~9 R# _+ N
1 Electrophoretic Techniques 19
1.1 The Principle of Electrophoresis and Some Methodological$ I  E- [3 u% w) O
Background 19
1.1.1 Free Flow Electrophoretic Methods 20
1.1.2 Gels for Electrophoretic Techniques 217 u9 R' D7 `, G6 x  a
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 22" l4 d, |4 S1 k! w7 Z# E( B& F- N

1.2.1 The Polyacrylamide Gel 22
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27* b, u4 o0 V6 x# k: g
1.2.3 Blue Native Electrophoresis 328 @. @9 g; ]5 P3 a
1.2.4 Cationic Detergent Electrophoresis 34
1.3 Blotting 35  ]* }6 f2 b8 f# V. ^# M% k$ ^3 I
1.3.1 Electrophoretic Transfer 36* i% S" U& u5 X  p; T  s
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38- V+ t' c3 R: T2 w) w  i
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 445 l* {3 w! Q# r. K0 k
1.4.3 Isoelectric Focusing in Proteomics 45
1.5 Two-dimensional Electrophoresis 53/ V  q: y; N5 b1 f2 f+ Q
1.5.1 Sample Preparation 53* X6 R  C" S1 A- ^/ A# @) R2 n0 a
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77; t& R3 x& Z2 m! s6 }  N3 o! H" a" I
1.5.4 Second Dimension: SDS Electrophoresis 100, V# a' I7 r# g  ?% G' Y
1.5.5 Detection of Protein Spots 119/ z- t. z  b# `. H- L0 m3 [( l( Q
1.6 Image Analysis 125
1.6.1 Image Acquisition 125
1.6.2 Image Analysis and Evaluation 129
1.6.3 Use of 2-D Electrophoresis Data 137
1.7 Spot Handling 137
1.7.1 Spot Picking 139
1.7.2 Protein Cleavage 141% ~5 U/ [( n, q; `4 j
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography& X, f3 _6 s% O: y2 b
Techniques 151" U" u0 u4 ]; H) y6 `$ d7 m
2.1.1 Ion Exchange Chromatography 153
2.1.2 Reversed Phase Chromatography 1628 K' x: f) v& p! H8 v! |& ~
2.1.3 Affinity Chromatography 1679 U( }; [% B' E+ {3 j+ j" ~4 M8 d
2.1.4 Gel Filtration 172
2.2 Strategic Approach and General Applicability 174
2.3 Liquid Chromatography Techniques and Applications in Proteome
Analysis 176! f; ~* Y' W9 V& \
2.3.1 Peptide Separation 176* _5 n- k; G( k0 `; P
2.3.2 2DLC Peptide Separation 179
2.3.3 Affinity Chromatography and LC-MS/MS 187
2.3.4 Protein Pre-fractionation 189) i$ ]. A* e: d" v  G
2.4 Practical Considerations and Application of LC-based Protein# Q8 T1 c& B2 a; I9 j
Pre-fractionation 194
2.4.1 Sample Extraction and Preparation 196
2.4.2 Experimental Setup 197; X5 H, Y( B0 b( j
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 198
Contents
2.4.4 Reversed Phase Chromatography and8 J( L8 W( }' h/ I$ H# H
Protein Pre-fractionation 2057 N- R/ x- g' \, ]) ^
2.4.5 Fraction Size and Number of Fractions 210; {9 E, b1 w1 _: K- ^
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 215
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 2189 J6 s! U4 o0 b/ Z% O
3.1.2 Electrospray Ionization 222& O; u9 h  d; Q6 u- _- R" _
3.2 Ion Separation 2251 N# V$ O0 b( V2 N8 n
3.2.1 Time-of-Flight Analyzer 225. U. i" _9 J4 f' G9 Z
3.2.2 Triple Quadrupole Analyzer 227
3.2.3 Quadrupole Ion Trap 2282 H8 I/ ]0 \9 w
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
3.2.7 Fourier Transform Ion Cyclotron 232
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233, J) I" `1 W  [
3.3.1 Peptide Mass Fingerprint 2348 G% Y2 I  U. H6 C% e
3.3.2 Peptide Mass Fingerprint Combined With Composition3 G$ c9 W1 k+ Q& T$ d- Y0 p
Information 237
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
Information 238
3.3.4 Tandem Mass Spectrometry 2427 x$ I" S$ ~0 o3 A/ O  t
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259
3.4.2 Affinity Chromatography 260
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 2633 W- F. ?- b6 h1 _, J' V8 y# [
3.5 Protein Quantification Using Mass Spectrometry 264' {( p! e: x% B4 ~" h( n
3.5.1 Stable Isotope Labeling Approaches 2645 ~8 @% ?9 k! V+ ~
3.5.2 Isotope-coded Affinity Tags 265
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 2669 y  B% u( K. i/ o
3.5.4 AQUA 267: z% N, M( o9 m/ [9 H
3.5.5 iTRAQ 267
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 271+ ^$ F8 P% C+ o1 o
3.6.1 Bottom up Approach 2711 W$ Z8 u2 I9 a' s# }" ~
3.6.2 Top down Approach 272
4 Functional Proteomics: Studies of Protein–Protein Interactions 273
4.1 Non-immunological Methods 273. S- [7 N) U5 @+ _  n  d1 b
4.1.1 Separation of Intact Multi-protein Complexes 273. i/ r# _  o( F; [: V/ ?
4.1.2 Probing with Interaction Partners 2733 }0 j4 n6 L9 g: O8 d* x
4.1.3 Surface Plasmon Resonance 274& o) n; m. J( M) A4 [; \( b* b) Y
4.2 Antibody-based Techniques 275
4.2.1 Western Blotting and Dot Blots 2754 _# ~) L, Y8 A0 Q1 k
4.2.2 Protein Microarrays 276+ G5 T- _& w$ ?% _* X+ D& W
Part II: Practical Manual of Proteome Analysis 2793 u+ Y: `8 @3 }$ z: S1 s
Equipment, Consumables, Reagents 2819 x" \3 y0 U3 A  R* h4 y$ ~
Step 1: Sample Preparation 287! y! q4 R2 p, k. t; o) `( Q- i
Step 2: Fluorescence Difference Gel Electrophoresis 299
Step 3: Isoelectric Focusing 309
Step 4: SDS Polyacrylamide Gel Electrophoresis 323! ~5 h1 K# F0 ^: t. w8 q. Z
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357! D" Y7 ^. R/ M* I: K$ S6 x
Step 6: Staining of Gels 361- u! n7 E: j8 |2 e+ v
Step 7: Image Analysis and Evaluation of DIGE Gels 373
Step 8: Spot Excision 3833 z% {; b) ^6 D, Z
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' l: r( ?# t# t* G* m
Step 13: MS Analysis 399
Step 14: Calibration of MALDI-ToF MS 403
Step 15: Preparing for a Database Search 407* \+ K' [: [: ]' W' K7 {
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+ b( k: a& b3 d  s% F! w# E
1.4 Staining 417* ?; q$ n4 B0 K: x: a6 M
1.5 DIGE Fluorescence Labeling 418
1.6 Results in 2-D Electrophoresis 4217 r5 \7 `& s! ?; G, n4 x
2 Mass Spectrometry 429" n& k5 A4 D- E3 u

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