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本帖最后由 细胞海洋 于 2010-6-28 11:09 编辑 6 M7 S% Q9 X# i5 R
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Proteomics in Practice! A) E) \4 h T4 Q- B$ |4 m
A Guide to Successful Experimental Design3 R' I2 S# K; B( B, ^
- C6 G$ A( }7 L) Y* ]- ZAbbreviations, Symbols, Units XV
" x6 c; ?8 e7 g8 vIntroduction 1# J" v# y; g" \- h. \' _
1 History 1
' m+ F4 h% n, B6 x: M; L2 Critical Points 80 q8 x4 u8 e% h- a# k! F
2.1 Challenges of the Protein Samples 8 A, N8 n$ u: T- K6 F) E
2.1 Challenges of the Analysis Systems 11
# `. \1 O- m5 W s! [( W6 c7 s3 Proteomics Strategies 12! N" U6 S8 ]3 H6 N" l; D) T
3.1 Proteome Mapping 12' T6 p: M3 t. H) A
3.2 Differential Analysis 12
7 z4 ]/ O+ G' e" s. C( z6 t# w, j3.3 Time Point Experiments 13
/ b0 ~" @/ `" Y& K! y3.4 Verification of Targets or Biomarkers 13# ^& p, z* T# d$ y8 m( `& A
3.5 Integration of Results into Biological Context 13
6 g- W' p- [$ F! f3.6 Systems Biology 13
' R( y3 r. p7 M7 j6 v; N- e- w$ Z5 G9 N4 Concept of Experimental Planning 14
& P# w0 I6 ~2 h! m4.1 Biological Replicates 14
$ w: @: L3 ^7 D( [0 \3 Y4.2 Pooling of Samples: Yes or No? 14
( l* h+ s& T }+ N& F4.3 Pre-fractionation of Samples: Yes or No? 145 d0 g" W1 f* y# j
4.4 Which is the Best Workflow to Start With? 15& W) a: k' z/ @- s, X6 O
Part I: Proteomics Technology
2 ?6 K! C+ Y, [- ]+ O5 v1 Electrophoretic Techniques 19/ |7 L8 T' d3 ?( ^: H2 A
1.1 The Principle of Electrophoresis and Some Methodological
6 |/ {- K7 }: G" N; sBackground 19
" m% U3 Y" p3 J) b& @5 L1.1.1 Free Flow Electrophoretic Methods 208 g, j: f v; g; D
1.1.2 Gels for Electrophoretic Techniques 21
2 V, ?. O* E. s# y* O1.1.3 Electroendosmosis Effects 21
( T9 h6 U3 J4 T ]( _ c1.2 Polyacrylamide Gel Electrophoresis 22
( ~3 N. n$ r. R1 q5 Q3 O5 H' Q0 Y7 D, S6 |; E8 b
1.2.1 The Polyacrylamide Gel 22
" I2 |2 I7 i4 E1.2.2 SDS Polyacrylamide Gel Electrophoresis 27
2 V1 e: o" G" _1.2.3 Blue Native Electrophoresis 32
: O0 \( b. _1 Z$ h1 s+ I/ L6 ^1.2.4 Cationic Detergent Electrophoresis 34
2 {& ~4 `+ a P3 [+ I1 f1.3 Blotting 35
6 u( f7 H. X: ^4 F1.3.1 Electrophoretic Transfer 36+ J7 _' `! v U! I5 x
1.3.2 Protein Detection on the Membrane 36
0 J4 ?5 T C6 x1.4 Isoelectric Focusing 38- U$ N% Y0 B; L" n. d1 E9 J
1.4.1 Theoretical Background 39
# R: I3 P O6 C( |4 @& ^1.4.2 Preparation of IEF Gels 44! L" x6 g8 ]! U7 ~& t" R
1.4.3 Isoelectric Focusing in Proteomics 45
+ \0 d4 s9 d; p: r" s4 H8 e8 @6 }6 X1.5 Two-dimensional Electrophoresis 53
1 f4 n5 J+ I) |( P* A1.5.1 Sample Preparation 53
4 x" H: |1 e/ Q, Y1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
( V4 a. z6 H3 _; o1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77. Y. k3 d! {9 i, V( c( [
1.5.4 Second Dimension: SDS Electrophoresis 100
* o# ~: X% B" ?+ x2 \* @* l1.5.5 Detection of Protein Spots 119
7 b" c. S. E$ Z% a+ o( c- O3 I) I1.6 Image Analysis 125' [& A$ f: a! Z* U
1.6.1 Image Acquisition 125: O' c1 e$ r0 a) d$ n) w. v
1.6.2 Image Analysis and Evaluation 129
7 | y, k) n* O* y* M3 ~8 v, j1.6.3 Use of 2-D Electrophoresis Data 137
, Z0 p9 P# l$ m( K1.7 Spot Handling 137+ {8 u3 u. Y# V8 m6 a
1.7.1 Spot Picking 1396 {+ [5 U7 T$ W. f, {( v
1.7.2 Protein Cleavage 141( z/ f, q' Y# j8 i0 a" Q
Liquid Chromatography Techniques 1511 T- D$ s6 Q" W, E
2.1 Basic Principles of Important Liquid Chromatography
5 M0 @, l$ s1 j9 ?Techniques 151
% {- U/ d0 R* O2.1.1 Ion Exchange Chromatography 1537 P* p7 K' F2 }( K$ N. d
2.1.2 Reversed Phase Chromatography 162
/ N2 t& ~4 b$ C* \2.1.3 Affinity Chromatography 167
' U' ~) q( ?6 H9 ~0 U2.1.4 Gel Filtration 1720 v: y4 c" J' Y% L3 ]
2.2 Strategic Approach and General Applicability 174$ X& Y C, a( H" O# ]6 C/ v. [' d
2.3 Liquid Chromatography Techniques and Applications in Proteome
* A, Q: X9 x7 s) k) l+ zAnalysis 176; h9 W. W8 c7 Q/ C
2.3.1 Peptide Separation 176
3 J% |0 U6 E3 U/ @6 ~9 @' w2.3.2 2DLC Peptide Separation 179
( p/ n: Y$ Q; E s; e2.3.3 Affinity Chromatography and LC-MS/MS 187
" X0 |# Q$ U& O2 g6 E$ T% j, w2.3.4 Protein Pre-fractionation 1892 T; e3 F" u- v" u) X! A
2.4 Practical Considerations and Application of LC-based Protein
4 z8 Q8 W8 X5 `9 f( [2 DPre-fractionation 194; u6 @+ m, g4 i3 p
2.4.1 Sample Extraction and Preparation 196
- |! R% m! U+ t3 |+ r; P' x9 ~2.4.2 Experimental Setup 1974 i1 ]8 |; ^" f1 |$ |4 C6 O9 e, M
2.4.3 Ion Exchange Chromatography and& R5 Y: f/ ]3 y5 P
Protein Pre-fractionation 198
5 `' F/ `; \! u& kContents, T& ~" f! {) Y' {
2.4.4 Reversed Phase Chromatography and+ W) A' K; f9 ]" X* I" ]' J
Protein Pre-fractionation 205
& I W& ~$ t/ \& `% Y, |. T2.4.5 Fraction Size and Number of Fractions 210( O" J) \$ R P; Q* _) J
2.5 Critical Review and Outlook 211
) A% D, H' ?* q2 P7 t& t/ m7 N3 Mass Spectrometry 215
( A% A$ N$ \3 u4 `; P; L s' w3.1 Ionization 218
* }, K+ o! S! Y$ S6 Y3.1.1 Matrix Assisted Laser Desorption Ionization 218
$ ]+ h! @& S5 S7 i. D, u3.1.2 Electrospray Ionization 222
3 E, ^$ w/ x2 q5 ?; e( {- ~! R3.2 Ion Separation 2251 Q9 l4 d& X1 ~
3.2.1 Time-of-Flight Analyzer 225
1 A$ ~+ g- F; I8 V8 w+ q3.2.2 Triple Quadrupole Analyzer 2273 a+ j& Q( N( t, {3 a g6 n- n
3.2.3 Quadrupole Ion Trap 228) _& n& r: }/ h" X7 Q: `2 N
3.2.4 Quadrupole Time-of-Flight 230+ r9 E9 r) M0 R6 S* d
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231
, y$ q/ R9 Y; ?1 o: v; x3.2.6 TOF/TOF Analyzer 231 @1 j3 W# z7 M- q& t
3.2.7 Fourier Transform Ion Cyclotron 232' e9 g* H9 {, W$ C( e* I
3.2.8 Orbitrap 233
! O0 R7 m. Q& h! M! u3.3 Generating MS Data for Protein Identification 2339 `1 ? V# N5 S" ~
3.3.1 Peptide Mass Fingerprint 2343 C5 E& j) G1 A. s: w
3.3.2 Peptide Mass Fingerprint Combined With Composition
0 _& V. @9 l0 o' @: ?! ~' \Information 237. ~4 T1 P' D/ B; d; E! h
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
4 r8 Z" S' g# h; g9 g$ } A2 SInformation 238
; k O0 ^3 l. k: y3.3.4 Tandem Mass Spectrometry 242
. y8 I1 H9 ?& P+ A3 R3.4 Protein Characterization 2588 i W. |7 j; }' ]4 i% J
3.4.1 Phosphorylation Analysis 259# r- K0 J7 x z; p/ d8 W
3.4.2 Affinity Chromatography 260
8 L( V' M4 K# d3.4.3 Chemical Derivatization 2614 A4 b$ C6 Z' t0 v K/ Z9 v9 y
3.4.4 Glycosylation 2632 t- }- Q8 U! D9 y, X2 |7 x
3.5 Protein Quantification Using Mass Spectrometry 264
" F% q1 n! w8 t/ r: b5 V6 ]3.5.1 Stable Isotope Labeling Approaches 264
$ t$ u4 f" o1 w3.5.2 Isotope-coded Affinity Tags 265
$ k+ B( Z L/ m$ B( S3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
g# X1 }" |4 L7 ~3.5.4 AQUA 267# l1 a9 J9 w* ]& u c3 \
3.5.5 iTRAQ 267" T; }8 ^1 S/ ]5 K' b% B; p
3.5.6 Non-labeling Software Approaches 2682 O M2 Y% T/ c( S, g! ^+ q
3.6 MS Strategies 271
! @+ o8 P: ~5 R% ~( e, w+ {; ?3.6.1 Bottom up Approach 271
( t; T8 s! p, e0 h3.6.2 Top down Approach 272
6 T' x# ]) S3 t! e4 Functional Proteomics: Studies of Protein–Protein Interactions 273
* w6 D% p9 [/ t8 F( }4.1 Non-immunological Methods 273
9 ]4 `- z5 f7 ~: `4.1.1 Separation of Intact Multi-protein Complexes 273) I. z. ]( N! O
4.1.2 Probing with Interaction Partners 273
" D( b8 S B+ w8 g7 e9 P$ G, c4.1.3 Surface Plasmon Resonance 2741 n4 h& V G8 l, O. w/ r$ j
4.2 Antibody-based Techniques 275# a+ i# f* G$ |' ~5 S8 O
4.2.1 Western Blotting and Dot Blots 275
# @, d$ b7 D' C; s+ `3 q4.2.2 Protein Microarrays 276. l2 ~% K9 ~5 Y* R) H2 h; Q, Y/ Q
Part II: Practical Manual of Proteome Analysis 2798 |3 I6 t* i) l! ^7 L8 ^! J
Equipment, Consumables, Reagents 281
, X" q. W3 U; o3 F' ZStep 1: Sample Preparation 2872 `9 _' o/ R# y2 {$ ? H: k6 x
Step 2: Fluorescence Difference Gel Electrophoresis 2996 f% f3 z7 h" C) l
Step 3: Isoelectric Focusing 309& n( i& Y- T6 b+ g
Step 4: SDS Polyacrylamide Gel Electrophoresis 3235 _8 q$ t" f8 h. O0 s0 N% U
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357% @$ h; Y7 [6 O4 n$ p- V
Step 6: Staining of Gels 361
; s% G$ g6 s2 T$ C7 tStep 7: Image Analysis and Evaluation of DIGE Gels 373( t+ \$ m. j9 ] v& `; Y
Step 8: Spot Excision 383
. I7 L% p& z. VStep 9: Sample Destaining 387( H% P) K6 [; ]1 {4 b- }* H
Step 10: Protein Digestion 3894 T0 L! y, a' I6 v
Step 11: Microscale Desalting and Concentrating of Sample 393
1 w! s) ] ]4 p! {9 `Step 12: Chemical Derivatization of the Peptide Digest 397
6 U# i7 u6 ?( Y3 t. kStep 13: MS Analysis 399
) {& F+ {& [+ v8 FStep 14: Calibration of MALDI-ToF MS 403
1 W- P6 o$ K" x0 }$ hStep 15: Preparing for a Database Search 407% h) ]1 D' I' H7 C( |8 D9 D
Part III: Trouble Shooting 4119 {! `8 j6 e" G% I0 w4 V2 J
1 Two-dimensional Electrophoresis 413
; a( ]& k% B+ Q1 M/ b- R6 ?% }1.1 Sample Preparation 4136 o- C$ t% L" o' }
1.2 Isoelectric focusing in IGPG strips 414% u5 I& s! [. A" n/ v E9 ^3 k
1.3 SDS PAGE 416( v1 x X8 v4 n5 e7 S! |# E3 w
1.4 Staining 417
& x* Y; A6 K' S1 z# W. Y! ~9 B1.5 DIGE Fluorescence Labeling 418
; m/ p9 @$ M$ U) T1 u5 D: I1.6 Results in 2-D Electrophoresis 421. t0 I6 @4 H+ i# l
2 Mass Spectrometry 429( Z, T# t" q9 {- c" I; ~" R
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发表于 2010-6-27 21:10
