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本帖最后由 细胞海洋 于 2010-6-28 11:09 编辑
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6 O/ x- M. W8 e4 a( |' R# iProteomics in Practice
5 G& H* Z9 T: i/ E @A Guide to Successful Experimental Design
4 J _* b G* f9 j0 I, @! o) p; _0 x& z8 h
Abbreviations, Symbols, Units XV
+ k- c4 ]5 [; {Introduction 1
4 @" R" M* g2 k' H1 History 12 m9 w( `) a2 Q# X+ i- Y
2 Critical Points 8: j2 M Y' v; \: w" y9 q3 C0 Y% P
2.1 Challenges of the Protein Samples 8! \7 g' O% K9 p. [ D& _* [( n1 O+ c9 F
2.1 Challenges of the Analysis Systems 11
- l* ]" N: ^ M1 c/ e* t3 E3 Proteomics Strategies 12
0 P5 b0 `( o% F( k6 h/ e4 Z q5 H8 a3.1 Proteome Mapping 12
3 c" \6 W; x! E( `1 W3.2 Differential Analysis 12- P% K5 c% W/ E% V
3.3 Time Point Experiments 13
8 n; R3 ^+ M+ s4 R3.4 Verification of Targets or Biomarkers 13
( y, B: U0 ^9 N7 r* N3.5 Integration of Results into Biological Context 13 B: a& R! `. V
3.6 Systems Biology 13
1 u& s; @, L0 ~2 L0 J1 Y. X4 Concept of Experimental Planning 14
" M3 S, F. J9 _6 P: S4.1 Biological Replicates 14
! z) C, t' j" g- K+ F9 \4.2 Pooling of Samples: Yes or No? 14+ i* p5 A% t) P f9 e6 h# Q
4.3 Pre-fractionation of Samples: Yes or No? 14& ]5 `7 U( P% D
4.4 Which is the Best Workflow to Start With? 15
9 d/ V3 X3 s% J Q3 B, \" f# RPart I: Proteomics Technology
; V# V0 H/ M( z% C* C" i4 j1 Electrophoretic Techniques 19) c* e; V; Y) ~# k, K2 }
1.1 The Principle of Electrophoresis and Some Methodological# P, X1 p- X% j1 w
Background 19. R U2 F/ q/ A7 s5 O8 z
1.1.1 Free Flow Electrophoretic Methods 205 V# v& j8 F9 X: o. ^, B8 k
1.1.2 Gels for Electrophoretic Techniques 21
0 S- x5 R% x# b/ A$ f1.1.3 Electroendosmosis Effects 21+ T3 |) q& L( {" y ?! [/ X9 u
1.2 Polyacrylamide Gel Electrophoresis 22
1 X% O: _- O2 {+ n( F5 e7 I+ E' V/ ?
& N- X5 K/ Y$ d3 i! P1.2.1 The Polyacrylamide Gel 222 b! O' D& [1 r: c8 I2 G. z7 Y, {
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27/ b2 d3 y' m$ q. K; J/ ]
1.2.3 Blue Native Electrophoresis 327 N9 F- _; l: n
1.2.4 Cationic Detergent Electrophoresis 34: z7 W+ w, [1 a ^. O/ F, ]6 |4 B9 ^
1.3 Blotting 355 H# `) C$ J( F, s4 f( ^: K9 ]$ Z7 A
1.3.1 Electrophoretic Transfer 36
( v/ f$ S9 B3 O% N3 V# w6 f# q, A1 h1.3.2 Protein Detection on the Membrane 36
9 X5 M, ?2 `$ \! A9 R( B4 M$ k1.4 Isoelectric Focusing 38' B: |' @0 c5 a5 g$ U& Z
1.4.1 Theoretical Background 397 Q( O& y# u# ]+ g% h
1.4.2 Preparation of IEF Gels 44
& {1 ?3 H' k- K1.4.3 Isoelectric Focusing in Proteomics 45
$ l% \ F8 z1 F* ]* n" l1.5 Two-dimensional Electrophoresis 530 X: c7 Y4 s" D
1.5.1 Sample Preparation 534 N$ w6 V3 D2 f, x2 q5 h% c
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
2 ~5 [ J3 C. I+ G: X, u1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77
{/ J6 M6 x: |1.5.4 Second Dimension: SDS Electrophoresis 100( J1 X" k* S' T$ |
1.5.5 Detection of Protein Spots 119
9 O2 R8 p8 o( I" x! e4 c1.6 Image Analysis 125* w4 O q+ V2 g. k0 K
1.6.1 Image Acquisition 125
7 \+ s3 x3 r; D5 K; H1.6.2 Image Analysis and Evaluation 129
! q8 Y* o" C& W! c0 d3 ~1.6.3 Use of 2-D Electrophoresis Data 137
8 z" d: j( j$ c4 M, ]8 x1.7 Spot Handling 137
( W( N6 l! M4 ]' }8 {. `1.7.1 Spot Picking 139
9 w% k% N# b0 d) ?1.7.2 Protein Cleavage 141 A2 E: j- C! Q, r* m4 M
Liquid Chromatography Techniques 151- ^( M9 V) f( F j
2.1 Basic Principles of Important Liquid Chromatography
: X, |/ E; S1 a0 V c9 x# f' T# I' o) fTechniques 151
" ]8 \ b: k0 a6 ]+ S/ T! S: W2.1.1 Ion Exchange Chromatography 153/ O' n0 ^& X- l; V7 N
2.1.2 Reversed Phase Chromatography 162
* W- ?1 J4 K: r2.1.3 Affinity Chromatography 1674 D0 H8 z. r" n6 l; {0 r
2.1.4 Gel Filtration 172
2 f& v7 |+ r6 f; }- W6 c( K2.2 Strategic Approach and General Applicability 174' H- Y' t! V- _+ K3 r
2.3 Liquid Chromatography Techniques and Applications in Proteome3 V) t& Y& w1 S' f" G8 z
Analysis 176
% O, _: h' u4 q2 J2.3.1 Peptide Separation 176: I& K w3 m/ A8 V: F1 f( r" O6 g& n
2.3.2 2DLC Peptide Separation 179
5 m7 C# j8 S0 K0 ?2.3.3 Affinity Chromatography and LC-MS/MS 187; D5 v1 V' v4 C: t- s% s
2.3.4 Protein Pre-fractionation 1890 e2 o# C( P0 X. a% \3 w
2.4 Practical Considerations and Application of LC-based Protein
% [% v/ U3 P2 pPre-fractionation 194! l5 ~' D/ j. t7 V1 x: g
2.4.1 Sample Extraction and Preparation 196
* m' W4 c4 c& ^* Z5 m" E) t2.4.2 Experimental Setup 1973 x4 v# r4 a5 }$ K+ e
2.4.3 Ion Exchange Chromatography and
6 x# w- D" E: `+ O! _- Q4 R9 aProtein Pre-fractionation 198
1 `& N; w3 b* P( S# nContents
K; @# y' @: p2 ~4 A4 F& x% D2.4.4 Reversed Phase Chromatography and
+ f: x9 c' y4 TProtein Pre-fractionation 2053 x, r4 v; L, G
2.4.5 Fraction Size and Number of Fractions 210$ g6 l) ?7 F/ |0 W, C( P
2.5 Critical Review and Outlook 211! H( Q: q; p" v1 I2 |9 z, H
3 Mass Spectrometry 215& t0 J) ^& D% s) d: z
3.1 Ionization 2188 \" r- T* O' D' F
3.1.1 Matrix Assisted Laser Desorption Ionization 218
; m, v5 D+ ^1 j$ W# w3 w8 T3.1.2 Electrospray Ionization 222
& b% @! F& G2 j3.2 Ion Separation 225: [$ \! p6 s1 Y! }6 R
3.2.1 Time-of-Flight Analyzer 2254 u& C; `# A2 w0 S, ]& u* _
3.2.2 Triple Quadrupole Analyzer 227
2 V F* z% p( K6 ?% _5 o- `3.2.3 Quadrupole Ion Trap 228
* c, z' N& k5 g: X) J6 o3.2.4 Quadrupole Time-of-Flight 230
) s+ h9 X% c i I3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 2315 S+ k* g3 M1 N5 O+ C: i
3.2.6 TOF/TOF Analyzer 2311 B+ M' m+ B4 R, d
3.2.7 Fourier Transform Ion Cyclotron 232
P+ [0 W( y7 l3.2.8 Orbitrap 233
6 |! v5 [3 b+ U6 s" ?3.3 Generating MS Data for Protein Identification 233
) l' z$ K7 I& H, B3 t9 T3.3.1 Peptide Mass Fingerprint 2348 V5 h5 S- t' q) T S$ d0 N
3.3.2 Peptide Mass Fingerprint Combined With Composition# e1 N" i- l, k7 j' G0 z
Information 2377 O% B4 J2 P" L1 g9 _2 W6 `
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
. Q/ V& Q# O$ b5 F! d5 ?9 ]Information 238
5 V3 D: s0 Z9 ^& p" h1 q# V3.3.4 Tandem Mass Spectrometry 242! @) ]" G6 C* m
3.4 Protein Characterization 258, I0 O+ b& l6 c }4 K& i9 ]' |
3.4.1 Phosphorylation Analysis 259
, m( ^; p I/ O; Y* Q* o3.4.2 Affinity Chromatography 260' u0 J$ j0 e) Z
3.4.3 Chemical Derivatization 2613 F9 Y3 q% @# I$ j
3.4.4 Glycosylation 263
2 ^5 M! b1 R' J+ k/ G3 o% ~/ g3.5 Protein Quantification Using Mass Spectrometry 264
2 o# v; [* u( y3.5.1 Stable Isotope Labeling Approaches 264
& Y7 s$ h! j0 J% Z0 }2 z3.5.2 Isotope-coded Affinity Tags 2659 ~) v/ z7 F% e! Y s
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
7 b. @8 P# @9 |3 n$ r" P3.5.4 AQUA 267: M% L9 o A& ?- ] d8 [. f, X
3.5.5 iTRAQ 267& s0 B% z5 {" Q9 d* k1 D
3.5.6 Non-labeling Software Approaches 268
9 W u! U8 r" f' B1 R3.6 MS Strategies 271% _% J7 Z& `( e% A/ k, W) K% A: d
3.6.1 Bottom up Approach 271/ \* t9 u5 N9 z9 ]
3.6.2 Top down Approach 272
" T5 G" f { ?. X& N' M( I6 |4 Functional Proteomics: Studies of Protein–Protein Interactions 273
& }9 b. Z6 d9 v* Y4.1 Non-immunological Methods 273
5 R F3 }# V, N- D; q+ x4.1.1 Separation of Intact Multi-protein Complexes 273& _7 M9 |% L# C: n
4.1.2 Probing with Interaction Partners 2736 O. V7 S) l8 l, t8 C+ V
4.1.3 Surface Plasmon Resonance 274
3 f; d1 ?3 T% R0 d3 U0 n4.2 Antibody-based Techniques 275
6 \' n9 {, {7 t/ x H; ]1 a4.2.1 Western Blotting and Dot Blots 275% C2 I: U( y0 l0 O: I5 [* {
4.2.2 Protein Microarrays 276
6 s! C3 H2 s- U0 ^! O; H, SPart II: Practical Manual of Proteome Analysis 279
: q+ Z2 ?, l; c4 T" QEquipment, Consumables, Reagents 281
& L; \9 O% s; YStep 1: Sample Preparation 2877 @/ x1 w* |; g1 i) A+ [; v
Step 2: Fluorescence Difference Gel Electrophoresis 299% b3 w$ A7 ?: t- u; _" g
Step 3: Isoelectric Focusing 309
/ W I5 G% A- w( {2 Q/ D. o; lStep 4: SDS Polyacrylamide Gel Electrophoresis 323) A& S' A& u* p3 ^9 ?
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
$ f$ u: w9 G: rStep 6: Staining of Gels 3618 c& X' r/ G8 y4 w$ W7 X
Step 7: Image Analysis and Evaluation of DIGE Gels 3739 B, z+ Z+ V/ C$ A1 l9 L
Step 8: Spot Excision 3839 A) Q" p! N5 k6 H
Step 9: Sample Destaining 387
0 C" c& @ |: u& k7 WStep 10: Protein Digestion 389
& v5 p9 W: H0 }" D+ r' h4 N4 [Step 11: Microscale Desalting and Concentrating of Sample 393" l0 I5 O! N, Q5 D) e1 V
Step 12: Chemical Derivatization of the Peptide Digest 397
! Q8 i# p" `: Q7 LStep 13: MS Analysis 399
0 Q, t5 x0 _- ?9 L' C RStep 14: Calibration of MALDI-ToF MS 4036 J9 g4 z1 \. X7 s) h6 x6 l" E! q& {
Step 15: Preparing for a Database Search 407
$ a5 S" r8 `$ o+ ^5 I- NPart III: Trouble Shooting 4111 O# c! Y1 O+ I! v% Q0 E5 }
1 Two-dimensional Electrophoresis 413
: \, X7 u0 I' x3 k. O- L* t1.1 Sample Preparation 413# [( x) U( O8 {! {# \
1.2 Isoelectric focusing in IGPG strips 414- o0 |# E& d1 I3 {4 o/ i; w1 j
1.3 SDS PAGE 416
6 r/ g! v: o M1 `1.4 Staining 417
9 i8 `0 Q4 Q: n Z1.5 DIGE Fluorescence Labeling 418
7 [! n2 |. Z i( l" b1.6 Results in 2-D Electrophoresis 421% C# L8 K% F5 r9 _/ x8 W- W
2 Mass Spectrometry 429
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