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Proteomics in Practice
A Guide to Successful Experimental Design' Z7 f4 i* Q: m9 t$ c% `
/ A/ e# l# W: ^9 T. |4 l
Abbreviations, Symbols, Units XV' o; m% I. s. g7 k7 Y
Introduction 1$ m) W8 @0 Z6 T
1 History 1$ I$ _) ^2 E( h
2 Critical Points 87 \. |  t1 O6 a& s# c5 _
2.1 Challenges of the Protein Samples 8
2.1 Challenges of the Analysis Systems 11
3 Proteomics Strategies 12
3.1 Proteome Mapping 12
3.2 Differential Analysis 123 ]/ H! c% [+ A+ y
3.3 Time Point Experiments 13
3.4 Verification of Targets or Biomarkers 13
3.5 Integration of Results into Biological Context 13
3.6 Systems Biology 13/ n4 r, i5 x4 U9 o
4 Concept of Experimental Planning 140 ]# M& j4 S; X
4.1 Biological Replicates 14. f" ]8 e( d; l8 P* v& ]
4.2 Pooling of Samples: Yes or No? 144 h( X) J3 s. ~: @
4.3 Pre-fractionation of Samples: Yes or No? 14
4.4 Which is the Best Workflow to Start With? 15+ i+ T1 ]$ b/ L* m5 T! D+ G" U
Part I: Proteomics Technology
1 Electrophoretic Techniques 19
1.1 The Principle of Electrophoresis and Some Methodological
Background 193 M" c, v- [0 }
1.1.1 Free Flow Electrophoretic Methods 20, {7 L+ d  ?4 F2 p0 s8 B* _" {
1.1.2 Gels for Electrophoretic Techniques 21
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 22

1.2.1 The Polyacrylamide Gel 22/ C- @, P! \, o7 |9 v# k3 d
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27
1.2.3 Blue Native Electrophoresis 32" Y, d7 T+ K6 l" _
1.2.4 Cationic Detergent Electrophoresis 34% ?% t; p3 Y  a  Z) i9 D8 _
1.3 Blotting 358 X; ?' N# B  k# ^- g: D, @1 p
1.3.1 Electrophoretic Transfer 36
1.3.2 Protein Detection on the Membrane 368 ]# z. D) _% X& k4 w
1.4 Isoelectric Focusing 38
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 445 O0 U; f- O) r* ]
1.4.3 Isoelectric Focusing in Proteomics 45
1.5 Two-dimensional Electrophoresis 53
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
1.5.4 Second Dimension: SDS Electrophoresis 1006 f4 \% a" k. F6 _
1.5.5 Detection of Protein Spots 1190 ]  |3 N4 p5 m! B& d
1.6 Image Analysis 125( X+ G! r! m3 ]9 P
1.6.1 Image Acquisition 125
1.6.2 Image Analysis and Evaluation 129
1.6.3 Use of 2-D Electrophoresis Data 137
1.7 Spot Handling 137
1.7.1 Spot Picking 139$ c" x( o0 q* Z/ ?, W
1.7.2 Protein Cleavage 141( U, b: o& N9 h. _$ i
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151
2.1.1 Ion Exchange Chromatography 153
2.1.2 Reversed Phase Chromatography 162, V: N# C5 E9 _: @1 A4 \+ w) {6 x
2.1.3 Affinity Chromatography 1671 o) e  X+ N- [# f$ W' m
2.1.4 Gel Filtration 1721 r+ |8 `) Q+ n9 \1 e: y
2.2 Strategic Approach and General Applicability 174
2.3 Liquid Chromatography Techniques and Applications in Proteome+ S5 S3 j9 C+ @! ^6 e1 g6 X- I- C
Analysis 1764 f) c  ?- o( ?+ z& _+ ?- D  @
2.3.1 Peptide Separation 176' O8 [& [) V5 D" w
2.3.2 2DLC Peptide Separation 179
2.3.3 Affinity Chromatography and LC-MS/MS 187" |- w# g2 c* H
2.3.4 Protein Pre-fractionation 189
2.4 Practical Considerations and Application of LC-based Protein% S( R) S8 c" t# @/ l
Pre-fractionation 194
2.4.1 Sample Extraction and Preparation 196
2.4.2 Experimental Setup 197% G: ]- J# x% {% i
2.4.3 Ion Exchange Chromatography and5 N- r- q. ?3 J" h+ j! G0 A6 M% }
Protein Pre-fractionation 198
Contents8 @4 q% m' T3 Q, g
2.4.4 Reversed Phase Chromatography and
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
3.1.1 Matrix Assisted Laser Desorption Ionization 218
3.1.2 Electrospray Ionization 2221 `: o( Z9 G) n, R# n7 o7 u
3.2 Ion Separation 225
3.2.1 Time-of-Flight Analyzer 225
3.2.2 Triple Quadrupole Analyzer 227
3.2.3 Quadrupole Ion Trap 228! }/ v  ]1 o$ m, p6 m  e1 h
3.2.4 Quadrupole Time-of-Flight 230
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231- x5 z! u& C3 ]* U8 w! p% Y" M
3.2.6 TOF/TOF Analyzer 2312 U7 ^& a/ B" f- ~8 {' o
3.2.7 Fourier Transform Ion Cyclotron 232: w/ L) r6 {' {: F/ [. p7 E
3.2.8 Orbitrap 233: E' e- G4 H; [. [  [6 K  N+ h
3.3 Generating MS Data for Protein Identification 2338 a' d3 r/ W1 J, N, p( [8 c
3.3.1 Peptide Mass Fingerprint 234
3.3.2 Peptide Mass Fingerprint Combined With Composition! p+ {" S, X! n/ ~9 |+ R
Information 237. b3 x7 t5 G; a7 b% @5 |: @
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence3 D, |0 k/ v$ m+ [6 E0 B9 R  s
Information 238
3.3.4 Tandem Mass Spectrometry 242
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259
3.4.2 Affinity Chromatography 260( x( F  b: w# K8 y2 r- R: V
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 263
3.5 Protein Quantification Using Mass Spectrometry 264$ ^( Y) t; y& M
3.5.1 Stable Isotope Labeling Approaches 264
3.5.2 Isotope-coded Affinity Tags 265; q9 K% ]1 h( Y$ x
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 267$ c$ E3 E5 q; O
3.5.5 iTRAQ 267; l! y( @  S! m7 x
3.5.6 Non-labeling Software Approaches 268. ]* U, Z& `8 v* x! E
3.6 MS Strategies 271
3.6.1 Bottom up Approach 271! {2 w# [; A  C7 s  C
3.6.2 Top down Approach 272
4 Functional Proteomics: Studies of Protein–Protein Interactions 273
4.1 Non-immunological Methods 273
4.1.1 Separation of Intact Multi-protein Complexes 273* z! L5 G: T" p# m
4.1.2 Probing with Interaction Partners 273
4.1.3 Surface Plasmon Resonance 274
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 2795 Y7 H+ }4 u4 ?% Z3 l4 w
Equipment, Consumables, Reagents 281# F2 c+ p7 V/ u/ F0 e$ }
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 299; z; V( O: u! j5 l
Step 3: Isoelectric Focusing 309
Step 4: SDS Polyacrylamide Gel Electrophoresis 323' v! o0 u# ?4 u9 u8 ]  }: i
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357/ Z, c1 l5 C. D. G/ f/ Q& S
Step 6: Staining of Gels 361
Step 7: Image Analysis and Evaluation of DIGE Gels 3739 i" L: J. ~+ ]  E: R# z( b# u
Step 8: Spot Excision 383
Step 9: Sample Destaining 387  u- r7 S9 N; f$ x
Step 10: Protein Digestion 389
Step 11: Microscale Desalting and Concentrating of Sample 393- M: _8 q3 k" Z' y3 ?2 ]6 x) s' z
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 407
Part III: Trouble Shooting 411
1 Two-dimensional Electrophoresis 413
1.1 Sample Preparation 413
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416$ q, Y# ^( L# A- X* a: I
1.4 Staining 417$ E  C6 _/ j! C# o# M0 o/ R
1.5 DIGE Fluorescence Labeling 4187 I3 R+ i0 I3 ~$ l
1.6 Results in 2-D Electrophoresis 4210 k- R: a* N* }& Y
2 Mass Spectrometry 429

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