PDF电子书：蛋白质组学最新版Introduction to Proteomics [Liebler[1].Humana.2002] - 经典PDF电子书下载区干细胞之家



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Proteomics in Practice
A Guide to Successful Experimental Design& n& R2 n9 u4 K( n
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Abbreviations, Symbols, Units XV- e- P- S- @9 J& D% Z( d0 C
Introduction 1
1 History 1. y5 l! C7 q' f
2 Critical Points 8+ H1 Y0 A4 C4 o# q  d
2.1 Challenges of the Protein Samples 83 V  W* H% `' U9 N2 c# k
2.1 Challenges of the Analysis Systems 11, c! g) e- K" l" o, _. A- h4 ~
3 Proteomics Strategies 12
3.1 Proteome Mapping 124 z9 \1 W) X+ n# G3 o9 f& c
3.2 Differential Analysis 12+ E5 x& a; c4 y- k
3.3 Time Point Experiments 13! J6 L$ j2 M5 X8 s2 a+ y) I4 X
3.4 Verification of Targets or Biomarkers 135 L7 h" C# ?! P2 n( ~' X
3.5 Integration of Results into Biological Context 13
3.6 Systems Biology 134 y, P, Z# T" f+ g% v, U  b: z7 u0 A
4 Concept of Experimental Planning 14. W6 S) [: X/ _
4.1 Biological Replicates 14" K& ~, i3 ~# |( p3 k) K7 M$ x; k
4.2 Pooling of Samples: Yes or No? 14
4.3 Pre-fractionation of Samples: Yes or No? 147 K( p8 d% R1 F( k
4.4 Which is the Best Workflow to Start With? 158 q0 B& Y- o! z; M' J6 n6 b; ~
Part I: Proteomics Technology
1 Electrophoretic Techniques 190 C1 T2 ]( N, S3 b- T
1.1 The Principle of Electrophoresis and Some Methodological1 G2 Q  c3 U$ T6 K
Background 19; F4 @8 W2 c- Y) ~( y
1.1.1 Free Flow Electrophoretic Methods 20
1.1.2 Gels for Electrophoretic Techniques 21) o# X2 w7 W* m5 c! F8 x& A7 w
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 22

1.2.1 The Polyacrylamide Gel 22
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27
1.2.3 Blue Native Electrophoresis 324 |% q2 p# S; T: @$ i5 r7 }) f& d- t3 N
1.2.4 Cationic Detergent Electrophoresis 342 |  C( |0 v5 }3 Z7 y
1.3 Blotting 35
1.3.1 Electrophoretic Transfer 36: `# h- B( u, @7 N/ j4 u0 s! n
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38) o! Z7 q8 S! W0 `
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 44
1.4.3 Isoelectric Focusing in Proteomics 45% ^9 p1 K+ ^' N3 A# f) U6 j) E
1.5 Two-dimensional Electrophoresis 53/ w/ i3 v1 I& X3 E( z
1.5.1 Sample Preparation 53" Q; p& C6 g$ j/ T0 w& ~; H# K
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77
1.5.4 Second Dimension: SDS Electrophoresis 100
1.5.5 Detection of Protein Spots 119  j2 T. W6 X- V& R/ o. i  K1 ]
1.6 Image Analysis 125
1.6.1 Image Acquisition 125
1.6.2 Image Analysis and Evaluation 129/ h" a6 ]  i1 V! A$ r& q
1.6.3 Use of 2-D Electrophoresis Data 1376 G* d" i8 l, C/ L$ {4 l) v7 e
1.7 Spot Handling 137( }4 [0 M5 f9 v9 I8 y3 ~/ J9 h* a
1.7.1 Spot Picking 1392 @( M+ j7 ]4 k! k' R
1.7.2 Protein Cleavage 141; U1 |7 U+ Q  C' u
Liquid Chromatography Techniques 151' L! ]4 q7 e; L: L% c) n) w5 |
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151
2.1.1 Ion Exchange Chromatography 153( D1 G4 Q* {( i
2.1.2 Reversed Phase Chromatography 162
2.1.3 Affinity Chromatography 167; X# ~7 m' R+ r0 \% ]) q& ^' N+ w4 u+ b
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
2.3.3 Affinity Chromatography and LC-MS/MS 187. p+ J% C/ b- `6 q0 o
2.3.4 Protein Pre-fractionation 189$ F3 }, E1 k/ i; d
2.4 Practical Considerations and Application of LC-based Protein. d1 f- o9 `) Q0 M3 x$ Y
Pre-fractionation 194
2.4.1 Sample Extraction and Preparation 1961 G: V3 d* @$ B" C' W! e' L4 L
2.4.2 Experimental Setup 197
2.4.3 Ion Exchange Chromatography and8 a0 Z) L+ g; C  ]
Protein Pre-fractionation 198
Contents1 I- \& K% l: Y4 i, G- W6 o
2.4.4 Reversed Phase Chromatography and
Protein Pre-fractionation 205$ y  N) T6 W0 y( P: j/ w, I! P* ~
2.4.5 Fraction Size and Number of Fractions 210
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 2152 [1 I) d  V* ?+ B' g
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 218
3.1.2 Electrospray Ionization 222
3.2 Ion Separation 225
3.2.1 Time-of-Flight Analyzer 225( e' C( Y$ _1 X1 X( @: z
3.2.2 Triple Quadrupole Analyzer 227
3.2.3 Quadrupole Ion Trap 228
3.2.4 Quadrupole Time-of-Flight 230
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231
3.2.6 TOF/TOF Analyzer 231
3.2.7 Fourier Transform Ion Cyclotron 232
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233' S6 a3 D2 Y7 Y3 e6 `9 u- L
3.3.1 Peptide Mass Fingerprint 2344 d, S* {6 Q- `; J: Y
3.3.2 Peptide Mass Fingerprint Combined With Composition2 `7 G2 u5 a7 o% ^& [5 K' y, l% P% ^
Information 237: M# T8 ]$ m; o/ F: S  T% Q6 f  z
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence% H; ]# a2 C  U5 k/ A  p5 w! q
Information 238; O% M5 W; [/ i
3.3.4 Tandem Mass Spectrometry 242* ^/ O! |7 L: g5 s5 F' M$ F
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259, g% Z2 a4 H) E. e( `0 t8 d
3.4.2 Affinity Chromatography 260
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 263
3.5 Protein Quantification Using Mass Spectrometry 264, P  [! v( b/ Q" x" q' M8 T
3.5.1 Stable Isotope Labeling Approaches 264
3.5.2 Isotope-coded Affinity Tags 265
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266% V. _- S3 @9 C, Y  |$ y
3.5.4 AQUA 267
3.5.5 iTRAQ 2674 }2 R0 [0 ?# ]7 w/ K7 O! {, ?+ _4 v
3.5.6 Non-labeling Software Approaches 2681 |' _, A( U3 U3 P  m0 }
3.6 MS Strategies 2718 V' m4 n" k" e+ f3 M
3.6.1 Bottom up Approach 271
3.6.2 Top down Approach 272* T/ |$ L! M0 O  L
4 Functional Proteomics: Studies of Protein–Protein Interactions 273! t! g6 d- w/ }& _! G- M
4.1 Non-immunological Methods 273
4.1.1 Separation of Intact Multi-protein Complexes 273' V& ?: s* k. _1 s" @/ E1 x" @
4.1.2 Probing with Interaction Partners 273
4.1.3 Surface Plasmon Resonance 274* U) q  ]" s9 ~3 z* n. y: [
4.2 Antibody-based Techniques 275/ ], {- b/ V7 n2 E. A6 y
4.2.1 Western Blotting and Dot Blots 2752 v5 y' @6 f9 H/ J# N. o' \
4.2.2 Protein Microarrays 2768 V  d3 \' z6 y+ H$ D
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 281+ S7 g: `# A2 q- E9 N- K
Step 1: Sample Preparation 2876 Z- k* }# C6 T) E/ K& y
Step 2: Fluorescence Difference Gel Electrophoresis 2992 p4 a5 H- X2 ?5 L- K: d8 f
Step 3: Isoelectric Focusing 309, W5 p# l: [7 A9 @% m7 X: T$ L
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357& S7 r0 \2 e# |/ W" \4 g
Step 6: Staining of Gels 361
Step 7: Image Analysis and Evaluation of DIGE Gels 373; C+ \; Q8 n+ [( k
Step 8: Spot Excision 383
Step 9: Sample Destaining 387. K) {3 }4 @+ z  U! f# Q- W( A, x8 K
Step 10: Protein Digestion 389
Step 11: Microscale Desalting and Concentrating of Sample 393$ h4 g7 N% F3 N3 p$ V& p7 ]  [
Step 12: Chemical Derivatization of the Peptide Digest 397/ _5 [9 V$ O9 [& {/ M' ?
Step 13: MS Analysis 399
Step 14: Calibration of MALDI-ToF MS 403
Step 15: Preparing for a Database Search 407- I* v* r  z7 b1 F4 c) g, ^
Part III: Trouble Shooting 411  M" n, A% k) G3 y8 b( B; p# x
1 Two-dimensional Electrophoresis 413) I$ V+ G* g# M. z
1.1 Sample Preparation 4136 S0 e8 Q3 B0 _
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416% B. T% }% h2 [/ u4 r& V
1.4 Staining 417
1.5 DIGE Fluorescence Labeling 418% k5 a( \3 ]- }3 V7 q- a6 a
1.6 Results in 2-D Electrophoresis 421
2 Mass Spectrometry 429

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