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Proteomics in Practice. P' ?6 I# z+ O
A Guide to Successful Experimental Design- S- f  e. p, a' x% `* s9 @: r% Q
3 j. m% _& s- Z2 s
Abbreviations, Symbols, Units XV& Z/ T! W' R" L; u, [
Introduction 1
1 History 1. `5 E- s+ F& \8 u# N" t
2 Critical Points 88 J% a  T# p1 ]0 ~' B" P, n
2.1 Challenges of the Protein Samples 8
2.1 Challenges of the Analysis Systems 11( X& M4 ^- K& y. @, g+ i% A
3 Proteomics Strategies 12
3.1 Proteome Mapping 12& `' k, Y" J8 k2 l5 k
3.2 Differential Analysis 12& I8 k' m% F4 p* Z7 ^
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 138 j# k; N  L1 `! x. c3 I+ z
4 Concept of Experimental Planning 14* i2 z$ F# E7 D0 c& h) B% ]7 @) F
4.1 Biological Replicates 14
4.2 Pooling of Samples: Yes or No? 146 ^( u6 R' \/ @& o, V* \0 d
4.3 Pre-fractionation of Samples: Yes or No? 14
4.4 Which is the Best Workflow to Start With? 15" \8 Y, a' S5 }+ ?4 i
Part I: Proteomics Technology# {9 Y0 P/ F7 S* T
1 Electrophoretic Techniques 19- z! d. H) _1 N8 t2 u  T
1.1 The Principle of Electrophoresis and Some Methodological
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" Q' I, i: N5 ]7 v8 v% E" J4 p
" b; [- x7 i  g8 e/ y$ C3 a
1.2.1 The Polyacrylamide Gel 226 L1 H0 B  v# I. Y
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27
1.2.3 Blue Native Electrophoresis 32: }& @* h7 i" \8 D- M& V2 s9 M
1.2.4 Cationic Detergent Electrophoresis 34! d5 H( {8 u+ C/ X9 l
1.3 Blotting 353 C0 N2 ^: }2 v
1.3.1 Electrophoretic Transfer 36
1.3.2 Protein Detection on the Membrane 36' T$ W: H6 x# ?' K/ ], R
1.4 Isoelectric Focusing 38, m6 M$ R6 g% `1 C! F
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 44$ |  @+ P3 \& n( U: z8 J+ `
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" Y2 ?. \: K7 W' a1 R0 A  v
1.5.4 Second Dimension: SDS Electrophoresis 100
1.5.5 Detection of Protein Spots 119
1.6 Image Analysis 125' d0 [. z: j8 Z0 |" D/ w8 e
1.6.1 Image Acquisition 125
1.6.2 Image Analysis and Evaluation 1290 w& a) M1 r( ^' ?; p% H; H5 [
1.6.3 Use of 2-D Electrophoresis Data 137
1.7 Spot Handling 137
1.7.1 Spot Picking 139
1.7.2 Protein Cleavage 1417 t: v; ^1 w8 ^4 }+ k# _1 Z; g
Liquid Chromatography Techniques 1516 r( }' j# X3 R) o% j% W
2.1 Basic Principles of Important Liquid Chromatography5 L8 I' K8 L# T+ a
Techniques 151
2.1.1 Ion Exchange Chromatography 153
2.1.2 Reversed Phase Chromatography 162; f5 J6 d5 q/ \+ Y
2.1.3 Affinity Chromatography 167
2.1.4 Gel Filtration 172
2.2 Strategic Approach and General Applicability 174# M0 p# L; z* P, e8 P0 d
2.3 Liquid Chromatography Techniques and Applications in Proteome
Analysis 176
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 179" l7 z- a9 r- Y1 A3 b& I
2.3.3 Affinity Chromatography and LC-MS/MS 187
2.3.4 Protein Pre-fractionation 1896 J. c) J8 m" f0 }3 c
2.4 Practical Considerations and Application of LC-based Protein, D  B: T) e$ T) h/ F' Z& o
Pre-fractionation 194
2.4.1 Sample Extraction and Preparation 196, b, r/ {- R: a/ X( ]. c0 h
2.4.2 Experimental Setup 1978 C- {$ ^/ l7 x
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 198
Contents, m  y6 A1 ]3 ~3 X
2.4.4 Reversed Phase Chromatography and
Protein Pre-fractionation 2051 G0 F9 y/ f# m- v2 S# r/ c
2.4.5 Fraction Size and Number of Fractions 210  p5 J; {' t& \! r7 i  k; r
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 215! i1 P- L' K6 E. i3 k
3.1 Ionization 218
3.1.1 Matrix Assisted Laser Desorption Ionization 2184 J. _" ]! x8 M( q5 f
3.1.2 Electrospray Ionization 222
3.2 Ion Separation 225, F) y, L1 }% H9 G! J9 C# X" O
3.2.1 Time-of-Flight Analyzer 225
3.2.2 Triple Quadrupole Analyzer 2270 i6 @  S: n. \6 Z0 [, R
3.2.3 Quadrupole Ion Trap 2280 Q. M2 v& G$ a% a* f/ I
3.2.4 Quadrupole Time-of-Flight 230
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231/ A- ]: ?" l  d" |8 V+ Q
3.2.6 TOF/TOF Analyzer 231: M$ {( |' m$ i+ P) s
3.2.7 Fourier Transform Ion Cyclotron 232
3.2.8 Orbitrap 233
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, G8 M2 h. F( v. |& m  S- y
Information 237
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence% \3 |+ F0 T& ^7 c" A) A
Information 2380 Z1 {0 E: P% r" p4 l$ ?% ?
3.3.4 Tandem Mass Spectrometry 242$ B; c; ?' r% A
3.4 Protein Characterization 2580 y8 K6 `0 ^$ l4 h
3.4.1 Phosphorylation Analysis 259
3.4.2 Affinity Chromatography 260$ W1 c8 @$ x5 Q  X
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 263' L; I% V7 F' r: `6 _4 _8 r& D
3.5 Protein Quantification Using Mass Spectrometry 264
3.5.1 Stable Isotope Labeling Approaches 264' _7 D, v3 D% q# b1 R, o
3.5.2 Isotope-coded Affinity Tags 265  g& p4 R5 f3 [
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266
3.5.4 AQUA 267. e, ~7 }# }1 i/ N8 s6 B
3.5.5 iTRAQ 267: G- _* G2 Z. S6 E" U# Z6 y! x
3.5.6 Non-labeling Software Approaches 2686 S, w: y; Q5 v( `" _4 ?7 j
3.6 MS Strategies 271
3.6.1 Bottom up Approach 271: [: f  w0 U% ^; W1 q$ g
3.6.2 Top down Approach 272
4 Functional Proteomics: Studies of Protein–Protein Interactions 2732 e. A* N( S9 k
4.1 Non-immunological Methods 273
4.1.1 Separation of Intact Multi-protein Complexes 273! @( {0 O5 {$ u
4.1.2 Probing with Interaction Partners 273& y  B; r: y& K! ~' ?* B; m9 s
4.1.3 Surface Plasmon Resonance 274
4.2 Antibody-based Techniques 275  \; x! J: [3 ]) E
4.2.1 Western Blotting and Dot Blots 275
4.2.2 Protein Microarrays 276
Part II: Practical Manual of Proteome Analysis 279$ `3 q; v, |0 }+ n- S
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 299
Step 3: Isoelectric Focusing 3094 B  G% G6 m3 T0 r/ g7 n2 L
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
Step 6: Staining of Gels 361) O" J* I# f! b( E: E
Step 7: Image Analysis and Evaluation of DIGE Gels 373
Step 8: Spot Excision 383
Step 9: Sample Destaining 387: [7 j" c# e- Z  p& A  }1 o
Step 10: Protein Digestion 389
Step 11: Microscale Desalting and Concentrating of Sample 393+ U% n* ?0 N* ~7 W2 k( Z
Step 12: Chemical Derivatization of the Peptide Digest 397
Step 13: MS Analysis 399! S4 e+ I! y% S& I
Step 14: Calibration of MALDI-ToF MS 403) K* y% |7 D1 H
Step 15: Preparing for a Database Search 407% `# W, Q$ o& I; B! v
Part III: Trouble Shooting 4113 D8 n5 W6 f' ~
1 Two-dimensional Electrophoresis 413
1.1 Sample Preparation 413+ ~( v/ i+ ^, f7 C4 S; ~( O$ v
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416
1.4 Staining 417
1.5 DIGE Fluorescence Labeling 4181 u) p, J: h  x% r
1.6 Results in 2-D Electrophoresis 421+ o, b" ?9 H! h5 a" d
2 Mass Spectrometry 4294 z: E; P. @# |( A; w
+ R4 u! y, ]4 h0 V/ S# c9 L9 P: @
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