PDF电子书：蛋白质组学最新版Introduction to Proteomics [Liebler[1].Humana.2002] - 经典PDF电子书下载区干细胞之家



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Proteomics in Practice
A Guide to Successful Experimental Design9 k7 ~* W* U/ Q- c

Abbreviations, Symbols, Units XV
Introduction 1
1 History 1) M( J+ q5 ~) ?5 i  t: ~) ]
2 Critical Points 8
2.1 Challenges of the Protein Samples 8
2.1 Challenges of the Analysis Systems 11+ W7 c/ Y2 q7 \2 ^) E
3 Proteomics Strategies 12
3.1 Proteome Mapping 12% L+ P/ H7 u4 C, x4 |* Q5 d* b- k
3.2 Differential Analysis 12
3.3 Time Point Experiments 135 H9 F; H/ v+ l* C
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 143 g7 i" P7 P! E3 x
4.2 Pooling of Samples: Yes or No? 147 A6 Y5 \- i/ R+ b" W
4.3 Pre-fractionation of Samples: Yes or No? 14, L# n" P: g- P4 o* P5 K# x* u: j
4.4 Which is the Best Workflow to Start With? 15% r+ o. w, _  q, t) M) j
Part I: Proteomics Technology
1 Electrophoretic Techniques 19
1.1 The Principle of Electrophoresis and Some Methodological. M: T! ^+ {3 x+ N' \  h: y
Background 193 b5 ~+ u. a. f; F. F8 l8 R4 q
1.1.1 Free Flow Electrophoretic Methods 200 X8 R( ]) L$ Y
1.1.2 Gels for Electrophoretic Techniques 21  A! W/ Q0 i8 Z' ?; K- G
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# P0 |; c+ m6 O" z. I
1.2.3 Blue Native Electrophoresis 32
1.2.4 Cationic Detergent Electrophoresis 34+ ]1 P/ f  l" V" P# G
1.3 Blotting 35
1.3.1 Electrophoretic Transfer 36  M& k& y4 _5 g- n/ L( E: k& A. m
1.3.2 Protein Detection on the Membrane 363 A) z' i" m, z& v4 u
1.4 Isoelectric Focusing 38* ]8 z! e: l5 `9 [& \3 z+ G. n
1.4.1 Theoretical Background 39: {/ R) p  z* h3 f# `2 }
1.4.2 Preparation of IEF Gels 440 E& C7 o" l6 S4 _0 x% {
1.4.3 Isoelectric Focusing in Proteomics 45
1.5 Two-dimensional Electrophoresis 53
1.5.1 Sample Preparation 53
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 686 t9 w' B/ ^7 q) ]+ n0 S( p3 C5 A
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 1198 o" ^8 H5 |. d' @) u1 [
1.6 Image Analysis 1258 V' y( @1 Z( `8 ?) ~3 j1 j4 P
1.6.1 Image Acquisition 125! G: h' y. Q: E
1.6.2 Image Analysis and Evaluation 129
1.6.3 Use of 2-D Electrophoresis Data 137: e2 R7 Y% g* ?* @
1.7 Spot Handling 137+ N8 \5 V3 K6 Y. a# d7 `1 Q8 z
1.7.1 Spot Picking 139: ^  Z- ?$ E/ V8 O. B1 L; A
1.7.2 Protein Cleavage 1411 |( H* k. \' D3 Q( k# @4 i) h- C, C
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography$ `6 J# L  K/ t: v6 G
Techniques 151
2.1.1 Ion Exchange Chromatography 153& y# j' N9 \. O" U3 O# _
2.1.2 Reversed Phase Chromatography 162
2.1.3 Affinity Chromatography 167: B+ O4 J% |" K% J; J3 u( m
2.1.4 Gel Filtration 172
2.2 Strategic Approach and General Applicability 174, r; q0 c" @5 p% a1 a
2.3 Liquid Chromatography Techniques and Applications in Proteome
Analysis 176
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 179: _) y2 O- Z0 v: N" E5 R
2.3.3 Affinity Chromatography and LC-MS/MS 187
2.3.4 Protein Pre-fractionation 189( q3 a/ g" j- E5 m2 |
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 194, D- M/ c$ X7 D4 M
2.4.1 Sample Extraction and Preparation 196
2.4.2 Experimental Setup 197% |4 a+ @, \6 h" I5 i: U+ z9 e# l/ n
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 1980 e. X2 D; Y% [
Contents
2.4.4 Reversed Phase Chromatography and* i5 v. ]* ~2 F8 h8 H( L
Protein Pre-fractionation 205
2.4.5 Fraction Size and Number of Fractions 210" K8 R- z& m  J% ?( ?4 a
2.5 Critical Review and Outlook 211; o$ r; x/ g- G5 d
3 Mass Spectrometry 215& {: n. p5 O. I
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 218# ^! B. s& u" {- ^3 L& D/ s% M1 }
3.1.2 Electrospray Ionization 222- M! x: _( t" L: G
3.2 Ion Separation 2255 I+ w1 B* f* r4 y8 E3 K$ K
3.2.1 Time-of-Flight Analyzer 225
3.2.2 Triple Quadrupole Analyzer 2272 R" a# t0 e/ \& e# ]  C
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
3.2.7 Fourier Transform Ion Cyclotron 2326 w6 T9 R, ^* W4 ^
3.2.8 Orbitrap 2330 B; `& h9 u9 S, Q+ f
3.3 Generating MS Data for Protein Identification 2336 K& g' }( u! v9 z& j, J
3.3.1 Peptide Mass Fingerprint 234
3.3.2 Peptide Mass Fingerprint Combined With Composition
Information 237. p. j- n  h  ?0 {$ Z8 @& Q6 e! W
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence+ ~7 ~7 `1 i0 W; [  @; U( U
Information 238
3.3.4 Tandem Mass Spectrometry 242
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259/ s. A8 N- U* {
3.4.2 Affinity Chromatography 2609 N7 u9 L0 D) ]; A' o9 Q% ^
3.4.3 Chemical Derivatization 261# D$ a' d1 B  T5 {+ r' X( v6 H- ~) u
3.4.4 Glycosylation 263
3.5 Protein Quantification Using Mass Spectrometry 264
3.5.1 Stable Isotope Labeling Approaches 264+ ]  s+ E, `5 V5 c6 T2 R
3.5.2 Isotope-coded Affinity Tags 265, Y: e) y" ]; {
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 267
3.5.5 iTRAQ 267
3.5.6 Non-labeling Software Approaches 2686 R; _5 [0 [1 ^5 h: G
3.6 MS Strategies 271
3.6.1 Bottom up Approach 271
3.6.2 Top down Approach 272- H8 n8 r; h  U& `- x( x! _4 }
4 Functional Proteomics: Studies of Protein–Protein Interactions 273
4.1 Non-immunological Methods 273
4.1.1 Separation of Intact Multi-protein Complexes 273' Y: e7 W+ d' S
4.1.2 Probing with Interaction Partners 273
4.1.3 Surface Plasmon Resonance 274
4.2 Antibody-based Techniques 275
4.2.1 Western Blotting and Dot Blots 2751 I, f& B3 J5 Y
4.2.2 Protein Microarrays 2767 B3 C+ @" d* B1 s( k9 S5 l
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287- }9 z% j0 _: f' E% `; V; M
Step 2: Fluorescence Difference Gel Electrophoresis 299/ \: E- [9 c0 \3 T8 J6 \$ g2 w
Step 3: Isoelectric Focusing 3091 w; q; I% i' W' y6 z
Step 4: SDS Polyacrylamide Gel Electrophoresis 323* ]* Z. E' h, {( _$ U8 @! H" Z
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
Step 6: Staining of Gels 3613 t: V: E8 r( A4 ?4 s9 T$ G
Step 7: Image Analysis and Evaluation of DIGE Gels 373: D  O- F! e8 K7 p
Step 8: Spot Excision 383
Step 9: Sample Destaining 387, ~* F) }' I$ f. P8 r3 K
Step 10: Protein Digestion 3897 y2 j" O1 Q: J9 V, j
Step 11: Microscale Desalting and Concentrating of Sample 393+ A7 f/ P* @. Z# ~: U
Step 12: Chemical Derivatization of the Peptide Digest 3975 s: v7 [" N- W5 [8 O. _& n+ `8 K" l
Step 13: MS Analysis 399
Step 14: Calibration of MALDI-ToF MS 403; d8 K* v7 e5 I! Q* X5 B
Step 15: Preparing for a Database Search 407% N7 f& a2 g+ ^: m0 i9 c, o. c
Part III: Trouble Shooting 411# W: w0 a3 e8 C" z
1 Two-dimensional Electrophoresis 413# p3 Y2 F( f  o9 G
1.1 Sample Preparation 413+ n3 A5 }/ N. F4 B  C9 K
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416
1.4 Staining 417
1.5 DIGE Fluorescence Labeling 418+ j9 N# x8 X( [  V+ z, w
1.6 Results in 2-D Electrophoresis 421
2 Mass Spectrometry 429

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