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Proteomics in Practice8 x  {( H" e( r& K6 F' e
A Guide to Successful Experimental Design. j7 q/ Y; @0 Y' Z4 M& b& a* p

Abbreviations, Symbols, Units XV( Q& t1 ]/ s4 E: `8 Y% [( o' Z1 {
Introduction 1
1 History 1
2 Critical Points 8" T/ ]0 l: n8 c5 G) h# O2 ?/ Y
2.1 Challenges of the Protein Samples 8
2.1 Challenges of the Analysis Systems 11. H1 k3 H6 t# o4 W
3 Proteomics Strategies 12
3.1 Proteome Mapping 12
3.2 Differential Analysis 127 \+ H- x6 F3 D, |
3.3 Time Point Experiments 13% n" \2 k# H# u0 V# Y2 [
3.4 Verification of Targets or Biomarkers 13
3.5 Integration of Results into Biological Context 13
3.6 Systems Biology 13- ]. U7 U8 x' M9 G
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' E% ~  r) Y) j- K! z) N8 o
4.4 Which is the Best Workflow to Start With? 159 e& g8 q0 u& j3 E1 w& ~
Part I: Proteomics Technology
1 Electrophoretic Techniques 198 r6 `& |% [, _$ A$ e
1.1 The Principle of Electrophoresis and Some Methodological. Q: f$ B( @2 w( D
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
2 C( J& ^6 P4 r
1.2.1 The Polyacrylamide Gel 22% M( k+ N  c; S' C+ {/ }
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27
1.2.3 Blue Native Electrophoresis 326 f! x1 V( X0 M/ I+ f
1.2.4 Cationic Detergent Electrophoresis 34
1.3 Blotting 35
1.3.1 Electrophoretic Transfer 36
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38
1.4.1 Theoretical Background 39( @4 t  b) U- E" Z3 ~& B# d' E  _
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 682 t3 T( ~# t7 h1 {/ W! S; W3 V
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
1.6 Image Analysis 125& m0 K$ E/ H( k' v- A. k
1.6.1 Image Acquisition 1252 R5 s! A& S- c/ \- e$ n
1.6.2 Image Analysis and Evaluation 129
1.6.3 Use of 2-D Electrophoresis Data 1372 w+ W( ]  Z; L  I% f
1.7 Spot Handling 137
1.7.1 Spot Picking 1397 W3 E! w% U0 N: X
1.7.2 Protein Cleavage 141( V1 i8 ^9 L/ a5 D8 f6 y8 D
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography# J2 C6 g. A0 g% ^
Techniques 151
2.1.1 Ion Exchange Chromatography 153  y/ _* L1 t' D& j* q
2.1.2 Reversed Phase Chromatography 162
2.1.3 Affinity Chromatography 167' |5 |# K* I; X/ w+ P
2.1.4 Gel Filtration 172. I6 H( e8 u& R5 l' _" H+ r
2.2 Strategic Approach and General Applicability 174$ y& L$ B! m8 X
2.3 Liquid Chromatography Techniques and Applications in Proteome
Analysis 176: Y/ o7 R/ n7 w# b
2.3.1 Peptide Separation 176
2.3.2 2DLC Peptide Separation 179
2.3.3 Affinity Chromatography and LC-MS/MS 187% d% s1 [4 d, D4 ]* X
2.3.4 Protein Pre-fractionation 189
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! y3 S7 ^3 m- K$ h4 I0 l7 j
2.4.3 Ion Exchange Chromatography and
Protein Pre-fractionation 198
Contents% Y% ?7 ]: d6 y/ B$ g8 j  Z
2.4.4 Reversed Phase Chromatography and1 K+ c6 n! j8 B, j  x. G9 u
Protein Pre-fractionation 205
2.4.5 Fraction Size and Number of Fractions 210. N4 b3 n$ O) G' ]0 V
2.5 Critical Review and Outlook 211
3 Mass Spectrometry 215
3.1 Ionization 218) m0 I$ O4 Y3 z' w. [
3.1.1 Matrix Assisted Laser Desorption Ionization 218; C8 r0 o1 M3 L  f$ I
3.1.2 Electrospray Ionization 2225 ^. @) \  w. H3 y# `" I5 R- n5 x/ r- e
3.2 Ion Separation 225: \$ z& v& z+ H
3.2.1 Time-of-Flight Analyzer 225+ B# y  {, I1 S- E
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; p8 c3 D- \8 B3 d
3.2.7 Fourier Transform Ion Cyclotron 232" F* t! l0 G1 T" ]% |
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233% H! W& I$ t  Y0 m) v4 ~. s' ]
3.3.1 Peptide Mass Fingerprint 234
3.3.2 Peptide Mass Fingerprint Combined With Composition
Information 2376 n+ z  G1 U5 o3 o0 s. D7 l
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
Information 238
3.3.4 Tandem Mass Spectrometry 242
3.4 Protein Characterization 258; d3 _6 U' v. \* s
3.4.1 Phosphorylation Analysis 259( Y  o! U4 p6 s* a* r! [
3.4.2 Affinity Chromatography 2605 [3 i; t5 ^. O/ t8 W, o, s$ \
3.4.3 Chemical Derivatization 261
3.4.4 Glycosylation 263& i' b4 {* u& u* y) z, O+ ^
3.5 Protein Quantification Using Mass Spectrometry 264
3.5.1 Stable Isotope Labeling Approaches 264
3.5.2 Isotope-coded Affinity Tags 265! u) b9 B) [1 D+ Z, `& o* _
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 2666 f- q. K0 I" j& a3 M
3.5.4 AQUA 267! q( x4 v& F5 D. {; ]
3.5.5 iTRAQ 267  ^: D2 A0 g7 j) p; v
3.5.6 Non-labeling Software Approaches 268
3.6 MS Strategies 271
3.6.1 Bottom up Approach 271% }/ W$ P  d( ?* b
3.6.2 Top down Approach 272) V) u+ Z4 b; L
4 Functional Proteomics: Studies of Protein–Protein Interactions 273
4.1 Non-immunological Methods 2731 b/ \0 u' _8 C  v
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: I8 d% S. Y/ C. [2 M
4.2 Antibody-based Techniques 275( \4 n8 {0 h$ G5 \( O
4.2.1 Western Blotting and Dot Blots 275$ H/ ~7 O; e- a# t5 K! l8 B( D! c
4.2.2 Protein Microarrays 276
Part II: Practical Manual of Proteome Analysis 279
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287
Step 2: Fluorescence Difference Gel Electrophoresis 299+ Q- _  H- e3 K& A
Step 3: Isoelectric Focusing 309) R- o8 h# {) S
Step 4: SDS Polyacrylamide Gel Electrophoresis 323! S# z* p% U) L' _- c: y
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357) h& W' o" O& }: c/ y+ i
Step 6: Staining of Gels 3614 [7 Y) f# @! F" J( C/ w
Step 7: Image Analysis and Evaluation of DIGE Gels 373' m6 X! ?# _' |+ u, j
Step 8: Spot Excision 383
Step 9: Sample Destaining 387. V: b. B0 T$ T* a' l; o
Step 10: Protein Digestion 389! ~  f* W( h7 E1 P) {( p+ Z
Step 11: Microscale Desalting and Concentrating of Sample 393/ ~( z/ ^5 w5 I. ~: ^" m- j
Step 12: Chemical Derivatization of the Peptide Digest 397. r& T% I* \1 B6 S0 ?) e; G- n
Step 13: MS Analysis 399
Step 14: Calibration of MALDI-ToF MS 403
Step 15: Preparing for a Database Search 407% \  O. M, t, G5 f; _) A+ l
Part III: Trouble Shooting 411+ |7 y' s9 I, x1 B+ Q# A; ^0 X
1 Two-dimensional Electrophoresis 413
1.1 Sample Preparation 413
1.2 Isoelectric focusing in IGPG strips 4144 \3 a+ A# G0 T8 |
1.3 SDS PAGE 4164 h- P1 K7 f  L! _6 {0 o( I
1.4 Staining 417. H" |" i' R$ C( f, J; S
1.5 DIGE Fluorescence Labeling 418) v* `. m# R! s
1.6 Results in 2-D Electrophoresis 421
2 Mass Spectrometry 429
; _- k( ~, `$ O$ v+ }- `' e$ O
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