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Proteomics in Practice$ K; Y  J  j, g/ h0 S* X
A Guide to Successful Experimental Design
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Abbreviations, Symbols, Units XV* n, q( u: T' ~2 u0 p9 x
Introduction 12 U% P0 g/ u+ \9 q7 b# o( y
1 History 1
2 Critical Points 8, L! a9 t% G3 A# l" L7 W
2.1 Challenges of the Protein Samples 85 {$ Q  K% w0 w$ O6 j+ Q9 I
2.1 Challenges of the Analysis Systems 11) n6 W- a) Z3 s9 \3 T
3 Proteomics Strategies 12
3.1 Proteome Mapping 12
3.2 Differential Analysis 12
3.3 Time Point Experiments 13
3.4 Verification of Targets or Biomarkers 13% W7 }4 M) X8 q9 y7 E' u7 B
3.5 Integration of Results into Biological Context 13
3.6 Systems Biology 13
4 Concept of Experimental Planning 14) K1 p* K$ w4 d! b
4.1 Biological Replicates 14
4.2 Pooling of Samples: Yes or No? 14
4.3 Pre-fractionation of Samples: Yes or No? 141 b0 v. g, \# s0 v
4.4 Which is the Best Workflow to Start With? 15
Part I: Proteomics Technology
1 Electrophoretic Techniques 199 Z3 e( e8 k! f8 v9 f$ K' ]5 i. u
1.1 The Principle of Electrophoresis and Some Methodological) L0 O2 q0 \* ^* l2 m
Background 19
1.1.1 Free Flow Electrophoretic Methods 20
1.1.2 Gels for Electrophoretic Techniques 21
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 22
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1.2.1 The Polyacrylamide Gel 222 p6 C1 I! `8 e- S6 ]- ]
1.2.2 SDS Polyacrylamide Gel Electrophoresis 273 X' C& c; J4 X# y/ E! l: e1 T; G  M
1.2.3 Blue Native Electrophoresis 32
1.2.4 Cationic Detergent Electrophoresis 34
1.3 Blotting 35
1.3.1 Electrophoretic Transfer 367 f) k8 b4 q- U- u
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 44
1.4.3 Isoelectric Focusing in Proteomics 457 }) V$ N, R% f2 I5 R) {: H- }7 y
1.5 Two-dimensional Electrophoresis 531 M3 p' j, Z- `& J- j
1.5.1 Sample Preparation 53
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77+ G: g' N: l# @1 K# ]
1.5.4 Second Dimension: SDS Electrophoresis 100
1.5.5 Detection of Protein Spots 1192 T/ b4 {6 j4 f. m
1.6 Image Analysis 125
1.6.1 Image Acquisition 125, a+ G  U* w$ |) \2 k
1.6.2 Image Analysis and Evaluation 129
1.6.3 Use of 2-D Electrophoresis Data 137& }  U+ S* f# U8 S6 E4 a- q# x
1.7 Spot Handling 137; D- [0 u0 W/ G- U2 D
1.7.1 Spot Picking 139* N; T5 }" @$ S: t3 }
1.7.2 Protein Cleavage 141
Liquid Chromatography Techniques 1515 a* a; n4 _0 W1 `$ A" Y
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151
2.1.1 Ion Exchange Chromatography 153
2.1.2 Reversed Phase Chromatography 162
2.1.3 Affinity Chromatography 167
2.1.4 Gel Filtration 172
2.2 Strategic Approach and General Applicability 1746 W' H$ d% B* H( u+ A8 O
2.3 Liquid Chromatography Techniques and Applications in Proteome' n' E! n$ G8 _$ }
Analysis 176
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 1795 a, }0 O$ ~% C8 F, X8 s
2.3.3 Affinity Chromatography and LC-MS/MS 187
2.3.4 Protein Pre-fractionation 189& h+ y# V% N, C3 l' P: H; i, {
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 194
2.4.1 Sample Extraction and Preparation 196
2.4.2 Experimental Setup 197# e1 K/ ~6 e% M5 t& B2 D# l
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 198
Contents, ^: J9 z% l; A& G! e, c3 F; F* |
2.4.4 Reversed Phase Chromatography and- r1 n& ^% p4 {
Protein Pre-fractionation 205
2.4.5 Fraction Size and Number of Fractions 210
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 215
3.1 Ionization 218) I9 l; q4 P6 V
3.1.1 Matrix Assisted Laser Desorption Ionization 218
3.1.2 Electrospray Ionization 2223 \/ |# f" G9 I. [* X: W- o
3.2 Ion Separation 225: H* [2 V. ~7 @! W: Z, f' V
3.2.1 Time-of-Flight Analyzer 225
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 2328 c" y1 D+ X5 F5 a
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233
3.3.1 Peptide Mass Fingerprint 234; t+ d& u4 S  W
3.3.2 Peptide Mass Fingerprint Combined With Composition. w# e# J8 H: W7 l
Information 2373 Q9 c# M0 v) _+ L  I4 E
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
Information 238! ^% D7 ^: ]* L2 b: \3 z. X
3.3.4 Tandem Mass Spectrometry 242& U  s) f6 M  _) @, l
3.4 Protein Characterization 2587 Q) U: ^% Q; ]9 E
3.4.1 Phosphorylation Analysis 2598 R3 {6 {% c0 N2 {; N' ~
3.4.2 Affinity Chromatography 260
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 263. b5 d; ~8 @* F) }
3.5 Protein Quantification Using Mass Spectrometry 264
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- l* V% {8 e# B3 y
3.5.4 AQUA 267) K+ |8 [8 v, d. D7 M/ ^
3.5.5 iTRAQ 267
3.5.6 Non-labeling Software Approaches 268$ \" `; q! u2 Y  M
3.6 MS Strategies 271
3.6.1 Bottom up Approach 271
3.6.2 Top down Approach 272
4 Functional Proteomics: Studies of Protein–Protein Interactions 2735 P1 E+ D6 o" e! q
4.1 Non-immunological Methods 273- ]* o$ n6 \( R" S) c2 n3 {3 J% p
4.1.1 Separation of Intact Multi-protein Complexes 273
4.1.2 Probing with Interaction Partners 273
4.1.3 Surface Plasmon Resonance 274; i6 `& S8 {) C7 _2 m6 h0 t
4.2 Antibody-based Techniques 275
4.2.1 Western Blotting and Dot Blots 275
4.2.2 Protein Microarrays 276
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 2813 Y- B8 [- }- j& w9 S" p
Step 1: Sample Preparation 287; [/ D6 e! R( L" x2 _* s% a
Step 2: Fluorescence Difference Gel Electrophoresis 299" c( R$ P) m1 M: M4 ^
Step 3: Isoelectric Focusing 3095 q& m- _( k5 t1 E
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
Step 6: Staining of Gels 3619 P+ ?. q9 s/ `5 L/ C
Step 7: Image Analysis and Evaluation of DIGE Gels 3739 {+ U2 k8 h& l. s1 Q4 n7 i" a; R2 C$ H
Step 8: Spot Excision 383
Step 9: Sample Destaining 387
Step 10: Protein Digestion 389( O* Q' L4 I8 g8 W; U5 C; L( Y
Step 11: Microscale Desalting and Concentrating of Sample 393
Step 12: Chemical Derivatization of the Peptide Digest 397
Step 13: MS Analysis 399
Step 14: Calibration of MALDI-ToF MS 403
Step 15: Preparing for a Database Search 4074 R# J6 {, Y$ U5 Q
Part III: Trouble Shooting 411
1 Two-dimensional Electrophoresis 413% l9 q" H8 ^. R6 W$ p: }- E* o# D' V$ B
1.1 Sample Preparation 413# W0 g/ `* J( ]9 V" U
1.2 Isoelectric focusing in IGPG strips 414# x7 j: O  \/ T( n) j1 @( }4 S! a
1.3 SDS PAGE 416
1.4 Staining 4177 D) B# v) h  r  V
1.5 DIGE Fluorescence Labeling 418
1.6 Results in 2-D Electrophoresis 4216 g2 ^* d! v; j2 z: W3 Y" T1 Z
2 Mass Spectrometry 429
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