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本帖最后由细胞海洋于 2010-6-28 11:09 编辑 6 J& J: G% e( o6 Y0 l
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Proteomics in Practice) o" o9 N2 Y9 g1 m, ]6 w0 Q( h
A Guide to Successful Experimental Design
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Abbreviations, Symbols, Units XV% N0 G! Z- R- ~9 N! i+ l5 L
Introduction 1
1 History 1
2 Critical Points 8
2.1 Challenges of the Protein Samples 87 D8 y9 B! k' z6 I/ b! d. w. [; E
2.1 Challenges of the Analysis Systems 119 G1 h1 @  p$ X3 q* L
3 Proteomics Strategies 12
3.1 Proteome Mapping 12
3.2 Differential Analysis 12' H2 @+ Y, ]4 F, M  n0 F/ J
3.3 Time Point Experiments 13
3.4 Verification of Targets or Biomarkers 13: Z/ M4 u2 F$ S6 @5 b
3.5 Integration of Results into Biological Context 13
3.6 Systems Biology 130 m, a1 T+ u- c" ~
4 Concept of Experimental Planning 14
4.1 Biological Replicates 14
4.2 Pooling of Samples: Yes or No? 14
4.3 Pre-fractionation of Samples: Yes or No? 14; r2 z7 E+ l. k  y
4.4 Which is the Best Workflow to Start With? 15
Part I: Proteomics Technology8 ~, _2 x  w" T7 ]
1 Electrophoretic Techniques 19
1.1 The Principle of Electrophoresis and Some Methodological
Background 196 A2 k  A  r' o: b4 i% J' O! ~
1.1.1 Free Flow Electrophoretic Methods 20
1.1.2 Gels for Electrophoretic Techniques 218 C8 P  i" U/ |, M! _( ], r
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 221 G# M& Z  i8 b5 |1 A6 z3 D

1.2.1 The Polyacrylamide Gel 22& W' V: {3 _% A3 o
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27
1.2.3 Blue Native Electrophoresis 327 N' V8 n# y7 I9 P' x
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 36
1.4 Isoelectric Focusing 388 F: Z1 c2 ?$ C5 Y1 _8 }& p
1.4.1 Theoretical Background 39' \6 L0 B, T9 ?" c
1.4.2 Preparation of IEF Gels 446 x0 W, Z8 N7 W0 N0 R
1.4.3 Isoelectric Focusing in Proteomics 45( e) \" D4 `' U! f4 G) D! k
1.5 Two-dimensional Electrophoresis 53
1.5.1 Sample Preparation 53
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77) }' X* S& u: |1 x. }1 v
1.5.4 Second Dimension: SDS Electrophoresis 1009 ~% a& C' ]$ _7 [& I8 D
1.5.5 Detection of Protein Spots 119
1.6 Image Analysis 125
1.6.1 Image Acquisition 125* X& W  s; R0 N: Y& E" D& b6 _
1.6.2 Image Analysis and Evaluation 129
1.6.3 Use of 2-D Electrophoresis Data 137
1.7 Spot Handling 1377 q5 t- m( r3 z4 _/ ~) p8 _9 W
1.7.1 Spot Picking 139) u  K7 u& a6 p5 t- M
1.7.2 Protein Cleavage 141- \" [& X! K/ |# x" j) c# r
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography4 o0 [5 e- N9 X- ~
Techniques 151' e& p8 \9 [* W* _# ^/ z, j/ e# X
2.1.1 Ion Exchange Chromatography 153, V; d' _) m9 e3 ?) G+ N1 f
2.1.2 Reversed Phase Chromatography 162+ O$ z* d7 T; @# H* {: @
2.1.3 Affinity Chromatography 167# g+ E! Z) Z0 y% q9 C
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
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 179- N2 V8 Q9 U. p# D6 M% h; x" U
2.3.3 Affinity Chromatography and LC-MS/MS 187
2.3.4 Protein Pre-fractionation 189
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 194
2.4.1 Sample Extraction and Preparation 196# y& Q- L9 e" V. v, h1 }3 \
2.4.2 Experimental Setup 197
2.4.3 Ion Exchange Chromatography and$ \2 F  D* B) z7 |' g0 `
Protein Pre-fractionation 198- t1 A7 S2 ~- w8 B1 }
Contents3 C/ q/ k8 C6 D# O
2.4.4 Reversed Phase Chromatography and
Protein Pre-fractionation 205# H9 R* Z/ ?9 F2 ^
2.4.5 Fraction Size and Number of Fractions 210
2.5 Critical Review and Outlook 2110 i! p: G- C& I: e6 \
3 Mass Spectrometry 215
3.1 Ionization 218  x( g+ o! E; w# J, _6 Z0 s9 \  F
3.1.1 Matrix Assisted Laser Desorption Ionization 218. r2 R- Q9 u5 Y* o9 P& O% D
3.1.2 Electrospray Ionization 2220 H1 s3 m# }4 x/ y- p& g
3.2 Ion Separation 2251 K" @; |% w6 c/ M9 t
3.2.1 Time-of-Flight Analyzer 225
3.2.2 Triple Quadrupole Analyzer 227! p8 t! T1 w  S$ D5 Y5 ~
3.2.3 Quadrupole Ion Trap 228$ i! O! l7 N/ k3 z
3.2.4 Quadrupole Time-of-Flight 230
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 2315 P5 z; s2 \+ C( D/ f, ]" i9 Q
3.2.6 TOF/TOF Analyzer 231
3.2.7 Fourier Transform Ion Cyclotron 232% t+ p7 m  t/ y
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233
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 Sequence6 M# A+ r' s1 R& G* \( F
Information 238
3.3.4 Tandem Mass Spectrometry 242
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 2593 h/ M; `0 o% K3 a# g
3.4.2 Affinity Chromatography 260( K! K! h/ e- v4 T6 ~( r
3.4.3 Chemical Derivatization 261: D0 W! z1 d; [/ {" x
3.4.4 Glycosylation 263
3.5 Protein Quantification Using Mass Spectrometry 264
3.5.1 Stable Isotope Labeling Approaches 264: G% I* I  `) U% z6 M/ \
3.5.2 Isotope-coded Affinity Tags 265: y: R* h$ K0 W. j" ^
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 267' z& Y& A1 n# s- J' i
3.5.5 iTRAQ 267; D0 A3 U: z8 S8 {
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 271: F+ K! ?$ Y2 r+ L$ `* V2 b4 W: l
3.6.1 Bottom up Approach 271
3.6.2 Top down Approach 272
4 Functional Proteomics: Studies of Protein–Protein Interactions 273* |% B- C; v# M8 i
4.1 Non-immunological Methods 273
4.1.1 Separation of Intact Multi-protein Complexes 273
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 2752 A3 s" F1 |3 V% Z7 e' ^8 Q
4.2.2 Protein Microarrays 2761 A; K8 G; q( v  u7 c7 s; G
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 299$ \" A1 W2 E. V6 G$ r
Step 3: Isoelectric Focusing 309
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
Step 6: Staining of Gels 361& J5 O- m3 M/ ?0 r2 l0 w( F2 ~' Q
Step 7: Image Analysis and Evaluation of DIGE Gels 373) T' X- r5 `* d- R9 T5 b1 S& _. R# ~3 P0 y
Step 8: Spot Excision 383( C7 N$ i" M2 h, f5 ^
Step 9: Sample Destaining 3870 [5 [/ T- b. g/ c$ [' @* y+ h# s
Step 10: Protein Digestion 389
Step 11: Microscale Desalting and Concentrating of Sample 393; B1 u. _$ U1 _" h8 m! B. e* b
Step 12: Chemical Derivatization of the Peptide Digest 397* j: G4 _- e) d6 q
Step 13: MS Analysis 3999 m% j5 x$ b- T/ I( f
Step 14: Calibration of MALDI-ToF MS 403/ z2 D# g' q$ A
Step 15: Preparing for a Database Search 407; {7 \, P0 T. _% E
Part III: Trouble Shooting 411
1 Two-dimensional Electrophoresis 413  `$ G$ S4 `% Q- y  k
1.1 Sample Preparation 413! e3 Z' x* @: z! l0 n* D9 [6 V
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 4163 M5 Q. |0 D" c" X: M! @$ ^7 d; p
1.4 Staining 417
1.5 DIGE Fluorescence Labeling 418
1.6 Results in 2-D Electrophoresis 421; O( M5 r+ O, O
2 Mass Spectrometry 429
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