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Proteomics in Practice8 |1 i: Q  t0 {/ ^5 m! s
A Guide to Successful Experimental Design: v& T8 M0 r' u: I6 O9 j) }% `

Abbreviations, Symbols, Units XV
Introduction 1+ U. d' \7 i, Z7 n8 S$ c. I
1 History 16 {  |* T. [0 y5 |
2 Critical Points 8+ g( I, O( E7 m. e
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! M1 F. c+ I5 g3 r5 C4 _3 b% H
3.2 Differential Analysis 12
3.3 Time Point Experiments 13- i: d9 H& ]2 B+ d3 F  i' \' D
3.4 Verification of Targets or Biomarkers 13
3.5 Integration of Results into Biological Context 137 W6 `8 X% ]# {
3.6 Systems Biology 13
4 Concept of Experimental Planning 14
4.1 Biological Replicates 14
4.2 Pooling of Samples: Yes or No? 14
4.3 Pre-fractionation of Samples: Yes or No? 14
4.4 Which is the Best Workflow to Start With? 15
Part I: Proteomics Technology
1 Electrophoretic Techniques 19
1.1 The Principle of Electrophoresis and Some Methodological3 k7 M, U2 W5 ?
Background 19
1.1.1 Free Flow Electrophoretic Methods 204 w8 C% b) B' p3 ~+ c7 [/ T
1.1.2 Gels for Electrophoretic Techniques 21/ x1 a* V* u) P3 g9 n, Y7 Q
1.1.3 Electroendosmosis Effects 21
1.2 Polyacrylamide Gel Electrophoresis 22
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1.2.1 The Polyacrylamide Gel 22, _: v. y1 Z1 f) b' j# O8 k4 p$ r1 `
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27
1.2.3 Blue Native Electrophoresis 32$ {3 s! e' n. C7 G
1.2.4 Cationic Detergent Electrophoresis 34
1.3 Blotting 35# o; k6 R$ E2 G- P
1.3.1 Electrophoretic Transfer 36' U3 W5 v1 U" p# Q! _
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38; r( ^7 o5 {) l. q
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 44
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 C2 q2 k9 F% u, q  ~' B0 T) ~- t" G
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 773 O7 ]4 i0 ^; P! S
1.5.4 Second Dimension: SDS Electrophoresis 100
1.5.5 Detection of Protein Spots 119
1.6 Image Analysis 125; S9 a9 V/ i$ I* k2 v3 [( m
1.6.1 Image Acquisition 125
1.6.2 Image Analysis and Evaluation 129$ h, e: w6 W3 ~
1.6.3 Use of 2-D Electrophoresis Data 137
1.7 Spot Handling 1373 k7 j4 f. P' {* S7 x- x* @+ w
1.7.1 Spot Picking 139" o- Y; B# Z2 J* U% I- y
1.7.2 Protein Cleavage 1418 l" y6 @. L" X( L$ K: Y
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151' T* k! `+ u1 w0 X" G
2.1.1 Ion Exchange Chromatography 153! i& B+ r& C: e9 I. N0 ?$ e! W
2.1.2 Reversed Phase Chromatography 162
2.1.3 Affinity Chromatography 1677 L/ v" I" h" p* O, p4 [
2.1.4 Gel Filtration 172# ^) D. S9 X5 t( X- g0 K
2.2 Strategic Approach and General Applicability 174, I" f8 d, b% U
2.3 Liquid Chromatography Techniques and Applications in Proteome
Analysis 176, [% A, ^2 Q7 M4 G# L: q: C
2.3.1 Peptide Separation 176+ i1 u3 Z" K' x1 u, f/ g
2.3.2 2DLC Peptide Separation 1794 j1 c+ [3 @: c- p0 @
2.3.3 Affinity Chromatography and LC-MS/MS 187/ w! {! [4 \' z. S9 _! ~
2.3.4 Protein Pre-fractionation 189
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 1943 `1 s# T5 L$ A( u! e+ v" H
2.4.1 Sample Extraction and Preparation 1965 E8 j$ S, G# t: C" k' X
2.4.2 Experimental Setup 197* E" }/ j) U( Y2 k0 _
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 198
Contents
2.4.4 Reversed Phase Chromatography and- F, ?5 u; S$ j- Q: b7 r  f5 W" f
Protein Pre-fractionation 205  f: b: O( K8 l' s
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: Y+ q( C8 Q' T+ l
3.1.1 Matrix Assisted Laser Desorption Ionization 218: x# K" `7 T( E5 w/ {& d3 U3 A* Q' E" \
3.1.2 Electrospray Ionization 2222 b! i  V0 |1 Z) T' X8 N
3.2 Ion Separation 2259 L; X8 S4 m& |  r5 |8 q* k$ |
3.2.1 Time-of-Flight Analyzer 225. w  h8 H* A  {/ `# R( M- r; t; d
3.2.2 Triple Quadrupole Analyzer 227+ h2 v& [6 h5 ^1 Z
3.2.3 Quadrupole Ion Trap 2287 Z" J# ?! O4 z7 }: ]8 H
3.2.4 Quadrupole Time-of-Flight 230
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 2312 s8 M0 B, h- z8 m/ x
3.2.6 TOF/TOF Analyzer 231# W. v3 T, k# K1 c. l/ s8 Y( |
3.2.7 Fourier Transform Ion Cyclotron 232
3.2.8 Orbitrap 2334 g  C" M# q2 ]9 J+ ~% I
3.3 Generating MS Data for Protein Identification 233
3.3.1 Peptide Mass Fingerprint 234
3.3.2 Peptide Mass Fingerprint Combined With Composition- i& U7 q8 _$ x1 H: n( V
Information 237
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
Information 238, q! p5 l. A. J+ |
3.3.4 Tandem Mass Spectrometry 242" |; x7 N0 s0 Q, Z
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259
3.4.2 Affinity Chromatography 260
3.4.3 Chemical Derivatization 2611 G& v5 c+ M# Z9 \6 k, }* ^
3.4.4 Glycosylation 263: I! A8 h6 Y+ D3 r
3.5 Protein Quantification Using Mass Spectrometry 2643 b/ n" `1 b7 {7 I
3.5.1 Stable Isotope Labeling Approaches 264
3.5.2 Isotope-coded Affinity Tags 2657 n' f# T+ y$ I" u$ _) K5 N* E( m" w% B
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 267+ h- f( l+ y: x8 X
3.5.5 iTRAQ 267
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 271
3.6.1 Bottom up Approach 2717 h. Y2 O+ M( S; F0 E: z5 q% F
3.6.2 Top down Approach 2722 f$ o8 r7 }3 k! `
4 Functional Proteomics: Studies of Protein–Protein Interactions 2730 g) w. l/ c" p% m' |" x+ o
4.1 Non-immunological Methods 273
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' Z$ Y. r+ n6 G( D( W' z
4.2 Antibody-based Techniques 275
4.2.1 Western Blotting and Dot Blots 275" j5 K9 n$ l  S! K8 r5 f% t( b
4.2.2 Protein Microarrays 2761 L% |9 s0 _* o+ C3 {4 X7 b
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287; H. L. R" g" [5 E4 m
Step 2: Fluorescence Difference Gel Electrophoresis 299! V. O. n& B% y; |) e) n% C5 k
Step 3: Isoelectric Focusing 309
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 3573 G" p& I+ n/ _* G% n$ F
Step 6: Staining of Gels 361
Step 7: Image Analysis and Evaluation of DIGE Gels 373, m+ w9 R! R# p2 F2 h) p$ s: G
Step 8: Spot Excision 383
Step 9: Sample Destaining 387
Step 10: Protein Digestion 389
Step 11: Microscale Desalting and Concentrating of Sample 393- F" U+ i$ A8 A. v5 e" ?
Step 12: Chemical Derivatization of the Peptide Digest 3976 A3 V0 k3 L: u( v' S( }, }- [
Step 13: MS Analysis 399; u( R0 W% O3 p/ X" h) u
Step 14: Calibration of MALDI-ToF MS 4036 S' m' w6 H  ?3 R- b$ ]: Y) }& w
Step 15: Preparing for a Database Search 407
Part III: Trouble Shooting 411
1 Two-dimensional Electrophoresis 4132 ?! m( H5 y+ v' N! X" L% [
1.1 Sample Preparation 4132 v8 i6 Z8 V# v* {; ]
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416- ^9 l$ a* D% K) e7 j
1.4 Staining 417* z$ h& C& }" z
1.5 DIGE Fluorescence Labeling 418& d+ Z" N- `' B. K" J
1.6 Results in 2-D Electrophoresis 4216 a7 G: ?5 o! Y! y+ T+ S
2 Mass Spectrometry 429
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