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本帖最后由细胞海洋于 2010-6-28 11:09 编辑 % d% A* j  {" d; t/ G
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Proteomics in Practice  p" `" E" K+ E$ y* z+ q" c
A Guide to Successful Experimental Design
  A7 c+ v0 M& U
Abbreviations, Symbols, Units XV+ s" o# a4 d$ j$ [) t9 A
Introduction 1) J7 w9 v* H, d; R* _) c' J
1 History 1
2 Critical Points 8
2.1 Challenges of the Protein Samples 8  K( _; d7 t: Q2 [
2.1 Challenges of the Analysis Systems 112 w3 }9 E% `0 l5 H! |+ N: {
3 Proteomics Strategies 12
3.1 Proteome Mapping 12/ |: w( h' q; v+ }; a
3.2 Differential Analysis 12
3.3 Time Point Experiments 135 ?9 m! o2 N; p0 l4 X
3.4 Verification of Targets or Biomarkers 13
3.5 Integration of Results into Biological Context 13, h$ h; @/ X* ]8 m  R
3.6 Systems Biology 13
4 Concept of Experimental Planning 14
4.1 Biological Replicates 149 ]2 m  V3 V6 x, M) n- C: e
4.2 Pooling of Samples: Yes or No? 14
4.3 Pre-fractionation of Samples: Yes or No? 14* H# a# `0 T/ v
4.4 Which is the Best Workflow to Start With? 158 @. \2 v0 f8 C* }* P
Part I: Proteomics Technology
1 Electrophoretic Techniques 199 \1 E2 e; ^# H
1.1 The Principle of Electrophoresis and Some Methodological
Background 199 S: F  [5 t5 A( e+ s
1.1.1 Free Flow Electrophoretic Methods 20; D' E: j0 f5 |! P' l3 j
1.1.2 Gels for Electrophoretic Techniques 21
1.1.3 Electroendosmosis Effects 21+ N6 Y- [) a3 L0 r8 d9 r( M* z
1.2 Polyacrylamide Gel Electrophoresis 22

1.2.1 The Polyacrylamide Gel 22
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27( h5 q0 }* Z$ v. |( }' [. Q
1.2.3 Blue Native Electrophoresis 320 B) u$ A5 B: J- X5 k; _2 C
1.2.4 Cationic Detergent Electrophoresis 34
1.3 Blotting 355 X& N" w7 }, ^. z0 p
1.3.1 Electrophoretic Transfer 36
1.3.2 Protein Detection on the Membrane 36; ?6 w$ j9 I9 s4 N4 D
1.4 Isoelectric Focusing 385 p: o6 L# o  E* V. j
1.4.1 Theoretical Background 390 G: n+ b1 w" C$ m
1.4.2 Preparation of IEF Gels 44
1.4.3 Isoelectric Focusing in Proteomics 45
1.5 Two-dimensional Electrophoresis 53
1.5.1 Sample Preparation 53  n3 n* m8 ~8 ?" Q
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68- l2 ]9 J' K6 W, H( V) s( @- G" m, |' O
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77
1.5.4 Second Dimension: SDS Electrophoresis 1009 q* U; w6 u/ A+ h1 l
1.5.5 Detection of Protein Spots 119% p2 T' S& q6 E
1.6 Image Analysis 125
1.6.1 Image Acquisition 125% `# P" ~+ e& ?% _! I
1.6.2 Image Analysis and Evaluation 129
1.6.3 Use of 2-D Electrophoresis Data 1379 }. M( x6 u. H  |  b8 s4 f" r
1.7 Spot Handling 137
1.7.1 Spot Picking 1392 w  b* R. d( [) }) _1 D% D9 t
1.7.2 Protein Cleavage 141/ v% t' Y9 f' L3 e/ s% k
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151/ U  i9 Q# R- S; H. X7 h
2.1.1 Ion Exchange Chromatography 153
2.1.2 Reversed Phase Chromatography 162
2.1.3 Affinity Chromatography 167
2.1.4 Gel Filtration 172
2.2 Strategic Approach and General Applicability 174
2.3 Liquid Chromatography Techniques and Applications in Proteome! ?) W* H$ p  {* }9 c9 Z3 x4 \
Analysis 176
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 179
2.3.3 Affinity Chromatography and LC-MS/MS 187- C! P1 y1 m: G8 s# S
2.3.4 Protein Pre-fractionation 189
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 194) y: R. Q5 i9 F$ w( e
2.4.1 Sample Extraction and Preparation 196+ B, E% c" u( I% g$ N  E, X
2.4.2 Experimental Setup 197$ o  S) z: p; D
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 198
Contents
2.4.4 Reversed Phase Chromatography and
Protein Pre-fractionation 205$ Y! E3 A  Q+ M. p1 J# d+ z0 R7 P
2.4.5 Fraction Size and Number of Fractions 2104 V* o$ b, o' g' {3 Q' h4 _/ X
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 215
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 218
3.1.2 Electrospray Ionization 222
3.2 Ion Separation 2256 t8 @$ A2 @9 ~- p/ r; t2 x
3.2.1 Time-of-Flight Analyzer 225
3.2.2 Triple Quadrupole Analyzer 227$ h* P0 h7 Q/ t, c
3.2.3 Quadrupole Ion Trap 228! H- W' a, ^8 W; I& N- v
3.2.4 Quadrupole Time-of-Flight 2301 I; {# `) F* w  n, ~4 x
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231
3.2.6 TOF/TOF Analyzer 231! B- x' H7 l. Q) M2 |$ H0 i, Z
3.2.7 Fourier Transform Ion Cyclotron 232  ]! Q7 n7 {/ ~" [% X  B- X
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233
3.3.1 Peptide Mass Fingerprint 234; S- d$ b6 F' W
3.3.2 Peptide Mass Fingerprint Combined With Composition
Information 237
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
Information 238
3.3.4 Tandem Mass Spectrometry 242
3.4 Protein Characterization 2585 T& R0 B0 ]9 \) I5 ~$ r
3.4.1 Phosphorylation Analysis 259. b+ E( Q8 [  ^  [$ m( `: K. F
3.4.2 Affinity Chromatography 260& d8 w  ^( i6 @$ x% c! H" v( H
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 2631 s  T, N1 j' }6 z- ]6 m0 Z7 i) p
3.5 Protein Quantification Using Mass Spectrometry 264
3.5.1 Stable Isotope Labeling Approaches 264
3.5.2 Isotope-coded Affinity Tags 265& k5 l. |% x% f! D
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 267" J% K. }* M; Y) W  F! w
3.5.5 iTRAQ 267
3.5.6 Non-labeling Software Approaches 268% P. ?& r! z3 c9 X6 V. ?6 q
3.6 MS Strategies 2716 H" Z6 E9 X* c# i, x% b
3.6.1 Bottom up Approach 271" b7 e% i1 Z" t0 o4 q; i8 i
3.6.2 Top down Approach 272: Q+ G& N5 A9 H8 g
4 Functional Proteomics: Studies of Protein–Protein Interactions 273
4.1 Non-immunological Methods 273  |# p* U3 t1 O; W
4.1.1 Separation of Intact Multi-protein Complexes 273
4.1.2 Probing with Interaction Partners 2739 A# W5 ?- S) X4 Y5 m" R" [) ^
4.1.3 Surface Plasmon Resonance 274
4.2 Antibody-based Techniques 275) [1 U, H! P7 S4 _! v: ^
4.2.1 Western Blotting and Dot Blots 2758 ?5 Z6 O# n, @0 p2 E' L% [
4.2.2 Protein Microarrays 276$ p3 T! N" X& |  b
Part II: Practical Manual of Proteome Analysis 279* b$ E5 k5 F' {- _+ ~; e  M
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 299. U! k$ K  M2 C4 J! a' b
Step 3: Isoelectric Focusing 309) |* j- W  e! V! l
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
Step 6: Staining of Gels 361
Step 7: Image Analysis and Evaluation of DIGE Gels 3730 q# h( R9 ~& q4 f- Q# p/ d
Step 8: Spot Excision 383
Step 9: Sample Destaining 387+ K7 p1 y5 {* y/ K! ?/ B
Step 10: Protein Digestion 389
Step 11: Microscale Desalting and Concentrating of Sample 3932 a" n1 S" G. ]; K
Step 12: Chemical Derivatization of the Peptide Digest 397
Step 13: MS Analysis 399
Step 14: Calibration of MALDI-ToF MS 403  _+ i' I& ]9 [2 }' y
Step 15: Preparing for a Database Search 407+ O' o1 n1 f5 m, K; [7 d: a5 m7 ~
Part III: Trouble Shooting 4114 o0 h' _& I6 @- S' i/ Q
1 Two-dimensional Electrophoresis 413
1.1 Sample Preparation 413# G) r, @4 s4 U6 s1 ^& N
1.2 Isoelectric focusing in IGPG strips 4142 H8 k) s+ Q$ k) e  Y1 a
1.3 SDS PAGE 4166 e6 C" y# r0 ]4 Z- z* X
1.4 Staining 4175 }* y4 N6 n+ B( `
1.5 DIGE Fluorescence Labeling 4189 Y( A5 |8 H# ~; _
1.6 Results in 2-D Electrophoresis 4212 ~; D7 I. p4 ^+ [) E5 x8 f
2 Mass Spectrometry 4292 k& A' q9 u5 ]. x' `% q
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