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本帖最后由 细胞海洋 于 2010-6-28 11:09 编辑 , ]! _( `0 l" `' I7 X. e
& d: w6 [0 Q+ D% \; hProteomics in Practice# D' s1 c! V) y$ }* m; Y% f- [
A Guide to Successful Experimental Design
; M! o z- F: |+ l. c; x8 P! ^1 H
$ |7 ?& e9 f$ NAbbreviations, Symbols, Units XV: m4 S2 R- W) ~8 P4 ^
Introduction 1
! s8 A4 B9 P2 F: x+ Q5 q& Z* c+ f1 History 1
& x& p4 {) b( E8 X! ^. s$ g9 L* Z7 ^2 Critical Points 8* X3 ^+ M& v# N+ J* A/ b
2.1 Challenges of the Protein Samples 8
]+ G; q2 f9 {1 u+ i2.1 Challenges of the Analysis Systems 115 q& j6 a$ J- S8 H b
3 Proteomics Strategies 12
6 G/ A9 {& [4 k. }* F3.1 Proteome Mapping 12! z C) K9 B) d
3.2 Differential Analysis 123 a9 M( e( ^$ U- m, O* B& ~1 u
3.3 Time Point Experiments 13
( }" M/ U9 l7 u( Q$ f5 a( y4 f7 G; O3.4 Verification of Targets or Biomarkers 13
( {2 L# \, G4 P! E. d3.5 Integration of Results into Biological Context 13) S7 w& l' F8 i1 L3 B
3.6 Systems Biology 13# D( ~9 k% W8 P$ C
4 Concept of Experimental Planning 14
' C' S- q# e$ I+ n7 f/ ~( a4.1 Biological Replicates 14* y V; p7 b9 }/ X
4.2 Pooling of Samples: Yes or No? 146 Q3 p7 k( x( q# y/ a, J. [ ^
4.3 Pre-fractionation of Samples: Yes or No? 14
, D, Y8 P! D) h% e3 {4.4 Which is the Best Workflow to Start With? 15
0 C/ v4 j' A! g1 V& t9 LPart I: Proteomics Technology
+ i" z+ B$ z/ A! k1 Electrophoretic Techniques 19+ i; Y$ P. W" k9 `$ ?: a
1.1 The Principle of Electrophoresis and Some Methodological
7 i! j: T# T( E, {5 x# BBackground 19( m, k4 b! F& Q* k( w$ N
1.1.1 Free Flow Electrophoretic Methods 20
& d# ?3 L9 z, D6 i# q; b& k2 Y1.1.2 Gels for Electrophoretic Techniques 214 I2 z+ V3 b/ y3 J: }. [) O
1.1.3 Electroendosmosis Effects 21
% r: I' Z: K' g" K l1.2 Polyacrylamide Gel Electrophoresis 22
: X( D, m) h! i# a0 b: ]& d5 b
1 I; _1 E; G1 R0 f0 I1.2.1 The Polyacrylamide Gel 22
+ _: j3 [ ~- ^$ L. r1.2.2 SDS Polyacrylamide Gel Electrophoresis 276 Q8 t* }! `1 d" G: x& { S
1.2.3 Blue Native Electrophoresis 32' x4 H" ^. R$ A# ]6 T' C& R
1.2.4 Cationic Detergent Electrophoresis 34
* \6 g' p2 P c1.3 Blotting 359 _/ s; Y4 u1 q) E0 b; G. m" k3 v
1.3.1 Electrophoretic Transfer 36
% w& [3 V. I$ ~7 r1.3.2 Protein Detection on the Membrane 36
0 q& C" @6 K1 b1.4 Isoelectric Focusing 38
; S2 o& B/ g# B, G* V1.4.1 Theoretical Background 39
- V. z& @1 |6 K6 f6 _1.4.2 Preparation of IEF Gels 440 G9 p$ d) Y) p+ V0 s) j1 N$ J
1.4.3 Isoelectric Focusing in Proteomics 45
' e- I+ Y+ R) c2 L$ V1.5 Two-dimensional Electrophoresis 53 M7 @4 {& A4 O3 [
1.5.1 Sample Preparation 532 k, O- f' E- h6 m7 R
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68' a8 X2 R9 a" U6 y* u+ N
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77
" e0 N/ e& ~- H; c/ V/ x1.5.4 Second Dimension: SDS Electrophoresis 100
4 j( r! s; M$ Z4 P. [0 f1.5.5 Detection of Protein Spots 119
/ [6 p8 h# |2 h6 x( [0 I1.6 Image Analysis 125
# V1 C* V- u% r, V% i1 J1.6.1 Image Acquisition 125
! }6 Y6 w+ L/ u9 ?1.6.2 Image Analysis and Evaluation 1292 u: u# w& g% I2 i7 i
1.6.3 Use of 2-D Electrophoresis Data 137
, |1 z* I/ ^9 e6 I0 d; e1.7 Spot Handling 137
; {' f9 d: R! z" X" \0 o+ S. j: J1.7.1 Spot Picking 139
! _+ ^1 R% k* |, _7 O& x/ ^4 o; V" h1.7.2 Protein Cleavage 141, f% e9 x$ v' B! r" f+ V- S9 [8 A
Liquid Chromatography Techniques 151
/ ?5 {8 Q* `4 U' f: z5 }2.1 Basic Principles of Important Liquid Chromatography8 |3 S/ | W9 i$ T5 j- d
Techniques 151
m/ C2 x+ V* ?/ D1 H s. ?2.1.1 Ion Exchange Chromatography 153
P" I- j" k `3 }% v2.1.2 Reversed Phase Chromatography 1625 B9 E3 O% f# f2 M1 \ I
2.1.3 Affinity Chromatography 1674 v% y6 P- ?% l2 E3 S& q! t
2.1.4 Gel Filtration 172
6 `) [4 z& K4 K" d9 I5 |+ g2.2 Strategic Approach and General Applicability 174& o" M* k3 E6 ~+ O/ u% i" }
2.3 Liquid Chromatography Techniques and Applications in Proteome7 P- h- k8 x( q7 `* M: E
Analysis 1764 z, S. [: E! R w( Z) n. n) g
2.3.1 Peptide Separation 176
2 a$ m! ~& A" X2.3.2 2DLC Peptide Separation 179. g, P/ c! A8 s5 M, l' E ]4 B2 ~
2.3.3 Affinity Chromatography and LC-MS/MS 187
0 V7 p( H7 Z5 P# `$ `2.3.4 Protein Pre-fractionation 189' `; F5 g$ Q2 w" \5 i+ o
2.4 Practical Considerations and Application of LC-based Protein3 m' _0 C7 R3 b% E# i" f* w
Pre-fractionation 194
# J8 z- |6 q" T6 e( }! R( F4 y2.4.1 Sample Extraction and Preparation 196
+ t) R- ] h5 M. U: A* F4 d h5 \5 P2.4.2 Experimental Setup 197 o4 U( ]5 ^* \8 g/ x) t
2.4.3 Ion Exchange Chromatography and
+ i! }. `% k N0 a- [2 I% IProtein Pre-fractionation 198, w% r+ c6 u; C* Q
Contents6 y8 l. G, ~# C3 K; j4 U0 O( H
2.4.4 Reversed Phase Chromatography and6 I# P" p3 C3 m- r& p
Protein Pre-fractionation 205
' D. j. S% ^) ^2.4.5 Fraction Size and Number of Fractions 2102 A1 a) i+ w& C, p
2.5 Critical Review and Outlook 211
" ^4 K; ^, Y2 r3 v. G; s3 Mass Spectrometry 215
6 j9 B; z9 l$ k( P1 h3.1 Ionization 218$ ?9 x2 c9 `4 f3 |0 Y. d
3.1.1 Matrix Assisted Laser Desorption Ionization 2186 X4 j5 R1 ]$ ~; t c. L2 L
3.1.2 Electrospray Ionization 2226 R: S! O. O4 v; U
3.2 Ion Separation 225$ K9 _& T, Q5 a3 ^' q7 F
3.2.1 Time-of-Flight Analyzer 225
* v! L2 n. e$ T# W8 s3.2.2 Triple Quadrupole Analyzer 227
2 b# z$ R3 c; U8 g4 t! k0 u6 u- n2 F3.2.3 Quadrupole Ion Trap 2282 ^- Q2 r% p; S9 N# ~. p
3.2.4 Quadrupole Time-of-Flight 230
% s8 |2 X# A$ X8 m/ ~# ^3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 2317 {- L7 ]. w+ F# m) {8 r8 F: q8 L4 d
3.2.6 TOF/TOF Analyzer 231
* M1 O+ E6 A0 m/ v( }" B' z8 V! X8 c3.2.7 Fourier Transform Ion Cyclotron 232
- |1 S" [+ K, R) Y- X% ?- }3.2.8 Orbitrap 233
, x* Q3 Q L6 n3.3 Generating MS Data for Protein Identification 233
$ H2 |+ e2 t- S" g+ h3.3.1 Peptide Mass Fingerprint 2346 z y+ A4 G d) }8 z) ^9 s
3.3.2 Peptide Mass Fingerprint Combined With Composition
* q; e2 u4 B1 \, Q ]* jInformation 237% D3 f# X+ E8 l! O' v6 M6 M
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence. N* k, K. i# s D- c
Information 238# V, l$ U/ N( M g
3.3.4 Tandem Mass Spectrometry 242" y' g0 `, U8 S! o# M
3.4 Protein Characterization 258) |9 `& Z7 ]: R. W: [1 t2 f6 W
3.4.1 Phosphorylation Analysis 259
, ~3 \* C4 _% y- j/ d3.4.2 Affinity Chromatography 260- ]: v6 K! Q$ p; y
3.4.3 Chemical Derivatization 261* R9 n0 D; m2 o- L! i5 X
3.4.4 Glycosylation 263
' w; J3 p" k5 J2 ?3 Q0 p3.5 Protein Quantification Using Mass Spectrometry 264
/ u, o- @( b' j5 r% }3 y3.5.1 Stable Isotope Labeling Approaches 264
7 g& H( e& q( K5 j6 m1 v3.5.2 Isotope-coded Affinity Tags 265" p/ |& e. M* L+ g" m" Q9 m' H
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266; d+ ?. B8 \% M
3.5.4 AQUA 267
, N3 a5 N5 O8 ^3.5.5 iTRAQ 267
3 {4 c* C a/ J. H7 C3.5.6 Non-labeling Software Approaches 268
" b1 M- X+ j/ B2 m/ `. A3.6 MS Strategies 271 Y! w& W3 Y# \/ v( t+ ]
3.6.1 Bottom up Approach 271
. o, v9 V+ d+ B% o8 S( N3 A3.6.2 Top down Approach 272+ @6 m! {3 `+ }0 P
4 Functional Proteomics: Studies of Protein–Protein Interactions 2731 K2 D" E q. f
4.1 Non-immunological Methods 273
& G- p* Z% j& B+ t; J. e4.1.1 Separation of Intact Multi-protein Complexes 273! ]9 p5 d* k: C0 Y" g+ e) L. y
4.1.2 Probing with Interaction Partners 273
( c2 n# K' @5 q+ l4.1.3 Surface Plasmon Resonance 274" k$ Z- {8 M- f$ e
4.2 Antibody-based Techniques 275
) z. v7 C$ h. q g, M! m4.2.1 Western Blotting and Dot Blots 275
/ [! i L" I1 K# a }4.2.2 Protein Microarrays 276
. a6 V; G( D' RPart II: Practical Manual of Proteome Analysis 279
7 {$ u! v& S( E6 k7 a6 dEquipment, Consumables, Reagents 281
! |2 l" w X% o6 F+ @+ x: VStep 1: Sample Preparation 2874 F; c/ u6 n1 G& k
Step 2: Fluorescence Difference Gel Electrophoresis 299! C* H, [! U/ ^, J% m' ~! j# b
Step 3: Isoelectric Focusing 3096 R9 y% o3 ?+ H9 `
Step 4: SDS Polyacrylamide Gel Electrophoresis 323% c! M3 C3 Q: d, @1 y# ~
Step 5: Scanning of Gels Containing Pre-labeled Proteins 3571 C( g* i- Q" P8 g/ n$ \( z+ O7 E# e
Step 6: Staining of Gels 3615 t f. n, `0 H5 E
Step 7: Image Analysis and Evaluation of DIGE Gels 373
, }: W4 L5 j' b, b' B6 Z2 {4 [Step 8: Spot Excision 383& z/ s: a; S( g+ y& X
Step 9: Sample Destaining 387
Z; x9 s! l. f0 x( u2 Z2 xStep 10: Protein Digestion 389
$ m5 _) {8 Z) nStep 11: Microscale Desalting and Concentrating of Sample 3930 ?: K9 f: f( P8 n5 C
Step 12: Chemical Derivatization of the Peptide Digest 397
7 Z) d$ Q1 [ ^7 F# i' @0 b5 E. AStep 13: MS Analysis 399* w" q& s' g9 r) a2 j2 r' h
Step 14: Calibration of MALDI-ToF MS 403$ i1 S# K: b8 l; b$ C* k
Step 15: Preparing for a Database Search 407
2 v/ A& }6 L) Y/ uPart III: Trouble Shooting 411
( E6 T# K, x8 y. P6 @( e* K8 T! q1 Two-dimensional Electrophoresis 413
+ H) x1 G/ D% {$ C4 E- R4 n7 q+ K1.1 Sample Preparation 4139 ]/ C4 y, {; X& \
1.2 Isoelectric focusing in IGPG strips 414
: l) k2 Y2 d) J1.3 SDS PAGE 416( H( k% d2 T" l# j: ^
1.4 Staining 417
" x9 g! e& j' B/ p5 I1.5 DIGE Fluorescence Labeling 4181 t0 g/ l, u& H/ a+ l$ |8 d
1.6 Results in 2-D Electrophoresis 4211 y: [4 e" s; Y4 J3 x5 k
2 Mass Spectrometry 429& M( F6 x3 w, p, W+ G8 h
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发表于 2010-6-27 21:10
