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Proteomics in Practice
A Guide to Successful Experimental Design

Abbreviations, Symbols, Units XV
Introduction 1
1 History 1
2 Critical Points 8
2.1 Challenges of the Protein Samples 8
2.1 Challenges of the Analysis Systems 11# ~7 @0 H0 }% d. {6 C) y
3 Proteomics Strategies 121 i' _# Q4 g( ^, j
3.1 Proteome Mapping 12
3.2 Differential Analysis 12
3.3 Time Point Experiments 13+ O& [+ H8 H% J) @7 h
3.4 Verification of Targets or Biomarkers 13
3.5 Integration of Results into Biological Context 13
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! X; M+ ]9 j2 Q3 ]5 v$ Z
4.4 Which is the Best Workflow to Start With? 155 }) {) W7 i3 y' L& o1 z3 E0 L7 m
Part I: Proteomics Technology
1 Electrophoretic Techniques 19
1.1 The Principle of Electrophoresis and Some Methodological
Background 19
1.1.1 Free Flow Electrophoretic Methods 20( A5 U0 V  V, y& y; Q/ d  j$ G; U
1.1.2 Gels for Electrophoretic Techniques 21
1.1.3 Electroendosmosis Effects 21& _- |. f' d/ X& c
1.2 Polyacrylamide Gel Electrophoresis 22+ m" l9 g) S5 _: K4 Z. j

1.2.1 The Polyacrylamide Gel 22
1.2.2 SDS Polyacrylamide Gel Electrophoresis 27& x4 W4 M& x, c" r- p* C
1.2.3 Blue Native Electrophoresis 32, {2 X/ ?) B8 y" W  L6 X, x
1.2.4 Cationic Detergent Electrophoresis 34% C1 A# V" c; H/ L
1.3 Blotting 35
1.3.1 Electrophoretic Transfer 36. ]$ C* L3 D) l. f; d4 P/ @
1.3.2 Protein Detection on the Membrane 36
1.4 Isoelectric Focusing 38! @! {$ H. ^/ q0 O2 f9 c
1.4.1 Theoretical Background 39
1.4.2 Preparation of IEF Gels 44, Q. R' p, P# F5 Y
1.4.3 Isoelectric Focusing in Proteomics 45
1.5 Two-dimensional Electrophoresis 53
1.5.1 Sample Preparation 53. a5 r: R3 C( I6 V+ k
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68/ O- z; P) W, @  n+ A  u) e
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
1.6.1 Image Acquisition 125+ V8 R0 o3 o+ L9 o# R& p$ L
1.6.2 Image Analysis and Evaluation 129/ F5 W  i, `& H9 ?( m% O  t, n  q
1.6.3 Use of 2-D Electrophoresis Data 137; f, z% s" t6 ], F( {; e
1.7 Spot Handling 137/ r1 A8 a; C. U- y
1.7.1 Spot Picking 139) J( H9 p1 \9 e# _- E: ?
1.7.2 Protein Cleavage 141
Liquid Chromatography Techniques 151
2.1 Basic Principles of Important Liquid Chromatography
Techniques 151# w* e5 l% l4 Z# Y4 |8 \* U
2.1.1 Ion Exchange Chromatography 153' |1 t( ^; W6 r# \0 Y: t
2.1.2 Reversed Phase Chromatography 1629 d; m) k/ ]0 P& y* L* W
2.1.3 Affinity Chromatography 167
2.1.4 Gel Filtration 172$ z0 H9 g9 ~6 N; o4 ]5 C8 m
2.2 Strategic Approach and General Applicability 1741 |  R( o9 K3 }7 ^5 P1 I  _
2.3 Liquid Chromatography Techniques and Applications in Proteome
Analysis 1766 n8 O) L7 c) o0 D, o! l
2.3.1 Peptide Separation 1761 G8 z1 m& V" \! D, G  l8 k. N
2.3.2 2DLC Peptide Separation 179
2.3.3 Affinity Chromatography and LC-MS/MS 187
2.3.4 Protein Pre-fractionation 189) x( m& h; r2 m1 ]- q0 p1 M  h
2.4 Practical Considerations and Application of LC-based Protein
Pre-fractionation 1947 E8 u( k8 _" R( z
2.4.1 Sample Extraction and Preparation 1964 ~7 o5 Z$ e6 N/ l: C# V5 o
2.4.2 Experimental Setup 1970 e9 E9 c# v+ |; g/ |4 Z
2.4.3 Ion Exchange Chromatography and& C+ y" z; r  U5 x- X. q
Protein Pre-fractionation 198
Contents
2.4.4 Reversed Phase Chromatography and
Protein Pre-fractionation 205- s1 b9 _1 I3 b' O1 }. H/ d
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 2184 W  @7 n0 J1 E
3.1.2 Electrospray Ionization 222
3.2 Ion Separation 225) O  t) j) q0 j8 O3 N
3.2.1 Time-of-Flight Analyzer 225( Y0 M0 q$ Q. l8 B/ j
3.2.2 Triple Quadrupole Analyzer 227( O, F4 k+ P8 @' k
3.2.3 Quadrupole Ion Trap 228
3.2.4 Quadrupole Time-of-Flight 230
3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 2310 s- h; Q# W, x
3.2.6 TOF/TOF Analyzer 231
3.2.7 Fourier Transform Ion Cyclotron 232+ X  f' T4 J) p: J; a4 ^( d- e
3.2.8 Orbitrap 233
3.3 Generating MS Data for Protein Identification 233" z; W5 ~. ^, l' n' s$ W) k
3.3.1 Peptide Mass Fingerprint 234
3.3.2 Peptide Mass Fingerprint Combined With Composition+ X6 l7 g/ R, O+ c, H' H! ?
Information 237! P- @, I# X' t4 u2 H) Z9 j% t
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence
Information 238; |$ X, n9 I0 B& X7 E' i3 G
3.3.4 Tandem Mass Spectrometry 242
3.4 Protein Characterization 258
3.4.1 Phosphorylation Analysis 259
3.4.2 Affinity Chromatography 260
3.4.3 Chemical Derivatization 2618 p4 a+ I- }8 s3 }4 D
3.4.4 Glycosylation 263/ o$ V1 n1 p7 N5 y8 ]* e
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
3.5.4 AQUA 267# t6 M( m/ t- p! A+ U
3.5.5 iTRAQ 2673 S4 l% x  w! Y! u' R! N0 f, \
3.5.6 Non-labeling Software Approaches 268! |4 ]) y* J4 V1 V  b
3.6 MS Strategies 271, {) F) ^8 n4 f4 [* Q" ^# J; X
3.6.1 Bottom up Approach 271& m2 ^  D  a- \( V6 r
3.6.2 Top down Approach 272
4 Functional Proteomics: Studies of Protein–Protein Interactions 2739 o( R2 B9 d3 Z" G
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
4.2 Antibody-based Techniques 275
4.2.1 Western Blotting and Dot Blots 275" l4 @' _/ _) K* r
4.2.2 Protein Microarrays 276
Part II: Practical Manual of Proteome Analysis 279: i0 c' p+ V+ H+ [3 q
Equipment, Consumables, Reagents 281
Step 1: Sample Preparation 287/ W& o% T( c! f2 r% g  O$ z4 R
Step 2: Fluorescence Difference Gel Electrophoresis 299, Z8 P3 \, [: X3 C: g' e
Step 3: Isoelectric Focusing 309) ~, g( c8 K- I
Step 4: SDS Polyacrylamide Gel Electrophoresis 323
Step 5: Scanning of Gels Containing Pre-labeled Proteins 357
Step 6: Staining of Gels 361
Step 7: Image Analysis and Evaluation of DIGE Gels 373" v, N( ^: B6 G7 }6 P) K+ M/ K/ v! e
Step 8: Spot Excision 383
Step 9: Sample Destaining 387+ P2 d1 [" m% M6 ~+ ]# e( [% w4 T6 Q
Step 10: Protein Digestion 389" z& O3 l3 d$ t7 T' n3 V- Q
Step 11: Microscale Desalting and Concentrating of Sample 393- q: c3 p8 e/ f! C
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& j) U; `( I- G  o
Part III: Trouble Shooting 411. ]+ a1 P( V* F: P
1 Two-dimensional Electrophoresis 413! m5 |0 v) V5 Z4 I1 A
1.1 Sample Preparation 413
1.2 Isoelectric focusing in IGPG strips 414
1.3 SDS PAGE 416
1.4 Staining 417
1.5 DIGE Fluorescence Labeling 418
1.6 Results in 2-D Electrophoresis 421" }, }+ H5 V! S! }
2 Mass Spectrometry 429
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