|
- 积分
- 1343
- 威望
- 1343
- 包包
- 387
|
本帖最后由 细胞海洋 于 2010-6-28 11:09 编辑
( p% A; ^! d1 A5 u# v# d, }6 \# V) h
Proteomics in Practice b: R. B5 W' @5 g0 b
A Guide to Successful Experimental Design5 l# }" X5 o! @9 S" v) U& E4 G
- F1 t+ X# a& d. h5 _- [Abbreviations, Symbols, Units XV
3 E( w! f7 H# dIntroduction 1
; f$ Y e6 S y) n7 _1 History 11 b: n* k4 |" w" S9 X" i1 M# I
2 Critical Points 8
0 h6 c& z1 W. v2.1 Challenges of the Protein Samples 8% L( F( F1 W9 x
2.1 Challenges of the Analysis Systems 11# H( ?* t0 ~7 u4 m( I3 w0 e
3 Proteomics Strategies 12) n7 Z6 J" h3 n8 v3 B2 B
3.1 Proteome Mapping 12
/ ?* x6 j7 i I' R3.2 Differential Analysis 12. }6 o- {5 G+ _; O9 k# D1 ?4 @
3.3 Time Point Experiments 13
2 V8 V7 x d8 m3.4 Verification of Targets or Biomarkers 13
4 S9 T! {: @, c3.5 Integration of Results into Biological Context 13
3 Y! C5 e0 @' `2 G D3.6 Systems Biology 13( v) O' _( k* h2 g' `
4 Concept of Experimental Planning 14
- M7 z. l# D. |! Q4.1 Biological Replicates 14
* Q [! @ V4 d7 Z5 f$ P4.2 Pooling of Samples: Yes or No? 14
" B8 w7 y$ q; x9 q. e* E2 K4.3 Pre-fractionation of Samples: Yes or No? 14
6 b7 m6 A) ~8 ^: f4.4 Which is the Best Workflow to Start With? 15
- x- x* y( a% ^' i+ jPart I: Proteomics Technology& i7 G/ k5 z: c. b$ x
1 Electrophoretic Techniques 19
# s+ O) J) i# w, g) w, b7 V" N1.1 The Principle of Electrophoresis and Some Methodological
1 u3 ~4 J8 Q# a; R ]Background 19
3 o+ a! ~ r' _) c1.1.1 Free Flow Electrophoretic Methods 20- Q/ h+ A! z$ A, r/ @& G# A3 V
1.1.2 Gels for Electrophoretic Techniques 21
9 c* p2 ^. | A) ]7 ]( G4 i1.1.3 Electroendosmosis Effects 21+ e0 A) u7 W2 r/ q7 w
1.2 Polyacrylamide Gel Electrophoresis 22
5 ^5 }% U! v$ i" j$ o o1 Y# v
4 m% D& w" M Q! ]6 G) B1.2.1 The Polyacrylamide Gel 22# ?1 L9 s( X: v. ?+ @/ I
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27 |* Z6 B, M4 o9 y1 x4 I$ G
1.2.3 Blue Native Electrophoresis 325 s' z; D+ [/ K2 v4 D( W1 G
1.2.4 Cationic Detergent Electrophoresis 34' E" P% M# `) i, M3 @ ` k
1.3 Blotting 350 i5 C) T9 E" w. L' R) ~' N
1.3.1 Electrophoretic Transfer 36
6 P9 l6 {" E9 t4 U5 c1.3.2 Protein Detection on the Membrane 368 i" O' S$ D4 g1 S. E
1.4 Isoelectric Focusing 389 u/ V6 b8 k5 V) M' l8 Y* R$ t- Y
1.4.1 Theoretical Background 39' g9 x( ? {" P% n7 \3 [
1.4.2 Preparation of IEF Gels 44
" G% g, Q- K5 H U2 I% w7 k) C1.4.3 Isoelectric Focusing in Proteomics 45
% ~% A: s9 `1 g8 Q. H7 R1 S; A9 C1.5 Two-dimensional Electrophoresis 53
% @, h! ?, X- P3 x0 a6 o) S1.5.1 Sample Preparation 53
) I G! M5 |: P6 l. b1 T0 J( S! a1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
) Q& p2 W# d5 W' G7 g0 Z1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 772 @! t6 q) {! c5 I. t, ^
1.5.4 Second Dimension: SDS Electrophoresis 100* X L& F8 e, y5 w; F3 {4 @- W
1.5.5 Detection of Protein Spots 119
2 \- ^3 y( e% }9 c5 h* f1.6 Image Analysis 125; L6 p/ a6 L& O( {
1.6.1 Image Acquisition 125/ J+ S/ P0 s, u) J8 i' F. X" e. `
1.6.2 Image Analysis and Evaluation 1292 ?& g7 e6 N( o2 x! b. p. N9 J
1.6.3 Use of 2-D Electrophoresis Data 137
9 ~5 J0 u8 x- A" J1.7 Spot Handling 137
+ `/ {/ l5 T6 d$ s/ |5 p$ w% M4 s1.7.1 Spot Picking 139" n" I$ y. j" e {/ n1 Z0 p/ Z
1.7.2 Protein Cleavage 141
% q3 _1 G# h2 k; z# @2 ~& d* M! O% dLiquid Chromatography Techniques 151
4 m+ I" Z* E- |+ e2.1 Basic Principles of Important Liquid Chromatography
6 A u" S1 E! UTechniques 151
# ^) X, a" h% L; [, v2.1.1 Ion Exchange Chromatography 153
) s) b7 R+ R; Q6 E- |/ @: j6 Y+ G2.1.2 Reversed Phase Chromatography 162( Y" @) c F! h
2.1.3 Affinity Chromatography 167% ^0 c5 ]7 a0 D; Y/ d$ O
2.1.4 Gel Filtration 172
; i0 U3 t* i0 t# f' E# K2.2 Strategic Approach and General Applicability 174; A. C" m* b- O8 _( k
2.3 Liquid Chromatography Techniques and Applications in Proteome) q5 L+ U/ Z4 R
Analysis 1760 C* x) t$ y1 d
2.3.1 Peptide Separation 176
- X8 ]4 k# r% W$ A: o* A2.3.2 2DLC Peptide Separation 179
; v. @; z3 F) L5 |# `. E( z! x9 ~2.3.3 Affinity Chromatography and LC-MS/MS 187
' I) k3 y$ v' W4 Y2.3.4 Protein Pre-fractionation 189
/ a& V2 B% `& f' H4 B' z8 `2 w2.4 Practical Considerations and Application of LC-based Protein1 W3 D! C7 Z6 {- V1 H4 O( f
Pre-fractionation 194
' |& K( W# ~7 K# I2.4.1 Sample Extraction and Preparation 196
- l5 f" B a0 K" U' f+ A2.4.2 Experimental Setup 197
+ u5 ?6 F' \' u) |5 [2.4.3 Ion Exchange Chromatography and1 y; F P0 [2 n. V) c
Protein Pre-fractionation 198) |$ v$ P& g2 m
Contents
1 E8 y2 p" S( q) C4 g9 Z! r# H2.4.4 Reversed Phase Chromatography and
3 h7 M5 C: y3 O! H$ G$ `, _! CProtein Pre-fractionation 205% m3 e/ Z1 k" V) V
2.4.5 Fraction Size and Number of Fractions 210
% v7 ~9 H, l2 e. s2 x2.5 Critical Review and Outlook 211- a7 e% D% q3 q: W
3 Mass Spectrometry 215
5 c* _9 k- G: p0 L1 [: v3.1 Ionization 218& L. W8 L+ z* z, q' Z
3.1.1 Matrix Assisted Laser Desorption Ionization 218
! D! }4 s" [/ C3.1.2 Electrospray Ionization 222- n, C/ l' N( n
3.2 Ion Separation 225
2 D; O% _1 C- F) X3.2.1 Time-of-Flight Analyzer 225
& ^' s2 o6 A' m- w7 S! O" v& V3.2.2 Triple Quadrupole Analyzer 227, X7 M7 E9 u7 F, z
3.2.3 Quadrupole Ion Trap 228+ q$ h1 \. U/ I: }9 m$ o
3.2.4 Quadrupole Time-of-Flight 230
3 H) N, j# l% D* ?8 g# G" y3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231
- Y/ R* Q* y2 Y3.2.6 TOF/TOF Analyzer 231& n& h/ U- L) S0 b. V
3.2.7 Fourier Transform Ion Cyclotron 232
0 @ S: H( d- e. v3.2.8 Orbitrap 2334 U, D1 D4 t* n( S: Q0 H' N
3.3 Generating MS Data for Protein Identification 2336 A7 |) ~: r/ H; u; O h* F
3.3.1 Peptide Mass Fingerprint 234
5 |* d: _. l0 W- A, [6 R3 {! B3.3.2 Peptide Mass Fingerprint Combined With Composition
( W9 R b, e8 I. B d0 I3 h, a1 I KInformation 237) l0 E' L/ `& \1 Y
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
& w9 ]) T$ g! M5 wInformation 238
3 W7 ]# E. S- h3.3.4 Tandem Mass Spectrometry 242
$ E8 H$ Z( H+ v3.4 Protein Characterization 2587 a) y$ _6 U' [' ]# v
3.4.1 Phosphorylation Analysis 259
. [4 z. s( T1 X( d2 g; m3.4.2 Affinity Chromatography 2604 P( B8 ?0 n% \3 F3 ^. P$ j) B
3.4.3 Chemical Derivatization 261
. a% v; O+ u1 m! a. Z3.4.4 Glycosylation 263' [: [4 w' j, \% ~
3.5 Protein Quantification Using Mass Spectrometry 2645 F) y5 k- k) M1 @/ _
3.5.1 Stable Isotope Labeling Approaches 264) ?& s+ C- w5 u0 c/ s$ W
3.5.2 Isotope-coded Affinity Tags 265
% h0 p/ O+ G6 a0 P+ L3 y" C, [3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266 \4 M: g4 T" t, a- V
3.5.4 AQUA 267
2 E* E A! s% ^0 O% c8 s3.5.5 iTRAQ 267
- J2 m8 e7 r% d I% ^; P8 Q3.5.6 Non-labeling Software Approaches 268
0 \) f5 }+ r4 G1 _8 _8 w. D3.6 MS Strategies 271
/ x4 i6 `* f; |- h: M% H5 _3 K3.6.1 Bottom up Approach 2718 V+ Y; i3 X- O: y; w) y
3.6.2 Top down Approach 272
) \3 y; Z2 B; r3 W) q4 Functional Proteomics: Studies of Protein–Protein Interactions 273% _6 ~6 [ y: O3 W0 d; Y
4.1 Non-immunological Methods 273
% t# l3 Q) b1 r4.1.1 Separation of Intact Multi-protein Complexes 273
' V; ]6 N0 t/ a/ p5 J' O4.1.2 Probing with Interaction Partners 273% k' I/ S/ O. X6 K5 }
4.1.3 Surface Plasmon Resonance 274
( u2 z1 i- r- `) T! Y# c4.2 Antibody-based Techniques 275
$ y+ _3 P1 I" k7 G/ i5 u6 ?4.2.1 Western Blotting and Dot Blots 2755 \' I) j/ r, T j
4.2.2 Protein Microarrays 276
; h' k, S( V8 I0 m! h& n4 QPart II: Practical Manual of Proteome Analysis 279
& ]1 W" @3 q" q' n8 uEquipment, Consumables, Reagents 2815 `! b5 I: Z2 b! x4 X
Step 1: Sample Preparation 2879 A1 C# O; z$ X9 ?$ |: Q g; e
Step 2: Fluorescence Difference Gel Electrophoresis 299
' g P& S6 f7 RStep 3: Isoelectric Focusing 309+ J) m4 w! }' [0 t
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
4 {' q: c3 C* s0 `; r/ ZStep 5: Scanning of Gels Containing Pre-labeled Proteins 357
L" q& n' y: p, jStep 6: Staining of Gels 361
5 s4 D& Q9 Q" z( [: u0 iStep 7: Image Analysis and Evaluation of DIGE Gels 373, d4 Z/ r" _5 U
Step 8: Spot Excision 383. |# ?# z2 z4 D7 c
Step 9: Sample Destaining 387 J# F5 R4 \+ f: o' B6 Z9 E
Step 10: Protein Digestion 389
; A x3 |& b' q* f! o5 lStep 11: Microscale Desalting and Concentrating of Sample 393
/ S9 G- o. w: V% `: ?( U8 aStep 12: Chemical Derivatization of the Peptide Digest 397
( Y- v- [! b! ^1 HStep 13: MS Analysis 3990 `6 W4 t1 j, [, E% \( W; {
Step 14: Calibration of MALDI-ToF MS 403! ^* _, q0 N% Q* V) W
Step 15: Preparing for a Database Search 407
) ~; O* m/ V) K! x2 FPart III: Trouble Shooting 411/ v6 _+ `8 M& P1 k6 c x8 }0 H
1 Two-dimensional Electrophoresis 413
& h% J/ x8 A+ X" |+ {7 v. C1.1 Sample Preparation 4135 N1 J: L8 z5 a6 b& [
1.2 Isoelectric focusing in IGPG strips 4147 }8 x1 m* i7 V" ~9 d: h' p
1.3 SDS PAGE 416, J0 L/ I, r7 i( y6 D4 D) D
1.4 Staining 4173 W- N3 E7 R# S# R- m& L
1.5 DIGE Fluorescence Labeling 418
" @# |5 K# s& q4 |" z, v! \+ P1.6 Results in 2-D Electrophoresis 4215 K6 {* \( d1 R* v m
2 Mass Spectrometry 429
! h" a6 a( A- I/ t- f6 T. q( k/ r) p2 H" p4 {! y
[hide][/hide] |
附件: 你需要登录才可以下载或查看附件。没有帐号？注册
-
总评分: 威望 + 5
包包 + 10
查看全部评分
|
发表于 2010-6-27 21:10
