Chemical Proteomics: Methods and Protocols

Cover......Page 1
Frontmatter......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 10
Part I: Introduction\r......Page 14
1. Introduction......Page 16
2. Global Chemoproteomic Profiling......Page 17
3. Targeted Chemoproteomics Approaches......Page 18
4. Impact of Experiment Design and Quantitative Read-Out for Target Identification......Page 20
References......Page 23
1. Introduction......Page 28
2. Chemical Proteomics Can Aid More Informed Selection and Validation of Targets......Page 29
3. Chemical Proteomics-Based Screening of Compound Libraries......Page 30
4. Chemical Proteomics for Drug Target Profiling......Page 31
References......Page 33
Part II: Small Molecules and Probe Design\r......Page 36
1. Introduction......Page 38
2.3. Cell Culture, Harvest, Lysis, and Protein Quantification......Page 40
2.7. Immunoblot......Page 41
3.1. Coupling to Solid Support......Page 42
3.2. LC-MS Analysis of the Coupling Reaction......Page 43
3.5. Affinity Pulldown......Page 45
3.6. One-Dimensional SDS-PAGE......Page 46
3.7. Silver Staining......Page 47
3.8. Immunoblot......Page 48
4. Notes......Page 49
References......Page 50
1. Introduction......Page 52
2.2. Cell-Culture, Cell-Lysis, and Microscopy......Page 54
2.4. Sample Preparation for SDS-PAGE and Western Blotting......Page 55
3.1.1. Preparation of the Modified FLAG ® -Coupled Bisindolylmaleimide-III (SMPC for Capturing of Protein Targets from Cell Lysates)......Page 56
3.1.2. Preparation of the Modified 5-FAM-TAT Coupled Bisindolylmaleimide (SMPCs for Capture of Protein Targets from Live Cells)......Page 57
3.2.1. Cell-Lysate Preparation......Page 59
3.2.3. Protein Preparation for Visualization and Western Blot Analysis......Page 60
3.3.1. Cell-Culture, Probe Incubation, Lysate Preparation, and Affinity Biochemistry......Page 63
4. Notes......Page 64
References......Page 67
1. Introduction......Page 68
2.1. Proteins and Staining Reagents......Page 69
3. Methods......Page 70
3.2. Native Gel Electrophoresis......Page 71
3.3. In-Gel Staining of the Dehydrogenase Subproteome Using CRAA......Page 73
3.5. Imaging the Gel......Page 74
4. Notes......Page 75
References......Page 76
1. Introduction......Page 78
2.1. Solid-Phase Synthesis of the Benzophenone Photoprobe from a PNA-Adenine Monomer......Page 79
2.3. Photolabeling......Page 80
3.1. Probe Design Considerations......Page 81
3.2. Solid-Phase Synthesis of the Benzophenone Photoprobe from a PNA-Adenine Monomer......Page 83
3.3. Jurkat Cell Culture and Preparation of Cytosolic Lysate......Page 84
3.5. SDS-Polyacrylamide Gel Electrophoresis and Western Blotting......Page 85
4. Notes......Page 86
References......Page 87
1. Introduction......Page 90
2.1. Cell Culture, HNE Treatment, and Derivatization......Page 91
2.3. Western Blot......Page 92
3. Methods......Page 93
3.1.2. Derivatization of HNE-Adducted Proteins and Streptavidin Affinity Capture......Page 97
3.1.3. Western Blotting......Page 101
3.1.4. In-Gel Trypsin Digestion and MS Analysis......Page 103
3.1.5. Database Searching......Page 104
3.2.1. aHNE Treatment and Biotinylation......Page 105
3.2.3. Photorelease of Adducted Plasma Peptides (Peptide Catch and Release)......Page 106
4. Notes......Page 107
References......Page 108
1. Introduction......Page 110
2.2. Capture Experiments and Controls......Page 113
2.4. Tryptic Digest of Proteins and Preparation of Peptides for LC-MS/MS......Page 114
3. Methods......Page 115
3.1. Preparation of E. coli DH5 a Cell Lysate......Page 119
3.2. Capture Assay, Competition Control, Pulldown, Competition Control of Pulldown, and Combined Capture Assay Plus Pulldown......Page 120
3.5. Tryptic Digest of Captured Proteins and Preparation of Peptides for LC-MS/MS......Page 122
3.6. NanoLC-MS/MS Analysis......Page 123
3.7. Peptide and Protein Identification via Automated Sequence Database Search......Page 124
4. Notes......Page 136
References......Page 137
Part III: Target Discovery and Target Validation\r......Page 140
1. Introduction......Page 142
2.2. SILAC Cell Culture and Preparation of Lysates......Page 143
3. Methods......Page 144
3.2. Preparing SILAC Cell Lysates for Pull-Down Experiments......Page 147
3.4. Gel separation and LC-MS Analysis......Page 148
3.5. Data Analysis......Page 149
4. Notes......Page 150
References......Page 152
1. Introduction......Page 154
2.1. Cell Lysis......Page 156
2.3. Competition Binding Experiment......Page 157
3.1. Cell Lysis......Page 158
3.2. Preparation of Affinity Matrix......Page 159
3.3. Competition Binding Experiment......Page 160
3.4. In-Gel Digestion......Page 161
3.5. Labeling of Tryptic Digests with Isobaric Mass Tags......Page 162
3.6. LC-MS/MS Analysis......Page 163
3.7. Data Analysis......Page 165
4. Notes......Page 166
References......Page 167
1. Introduction......Page 170
2.2. Preparation of the N -hydroxy­succinimide Active Ester of CRAA ( 21)......Page 172
3.1. Preparation of CRAA......Page 173
3.3. Synthesis of CRAA–Agarose Matrix......Page 174
3.5. Tandem MS Analysis to Identify Dehydrogenases in the Subproteome......Page 175
4. Notes......Page 176
References......Page 177
1. Introduction......Page 180
2.4. On-Column Isotopic Dimethyl Labeling......Page 182
2.7. Data Analysis......Page 183
3. Methods......Page 184
3.1.2. Tissue Lysis......Page 185
3.3. Dual-Protease In-Solution Digestion......Page 186
3.5. Strong Cation Exchange Fractionation......Page 187
3.7. Data Analysis......Page 188
5. Notes......Page 192
References......Page 194
1. Introduction......Page 196
2.1.1. Synthesis of Me 4 BodipyFL-NHS Ester......Page 200
2.1.3. Analytical Procedures......Page 201
2.2.3. Ex Vivo Profiling of Proteasome Subunit Activity in Murine Tissues......Page 202
3.1.1. Synthesis of Me 4 -BodipyFL-NHS Ester (Scheme  1)......Page 203
3.1.2. Synthesis of Me 4 BodipyFL-Ahx 3 Leu 3 VS......Page 206
3.2.1. In Vitro Profiling of Proteasome Activity in Cell Lysates......Page 209
3.2.2. Labeling of Active Proteasome Subunits in Living Cells......Page 210
3.2.4. Gel Electrophoresis and In-Gel Fluorescence Readout......Page 212
3.3. High-Throughput FACS-Based Proteasome Activity Assay......Page 213
4. Notes......Page 214
References......Page 215
1. Introduction......Page 218
2.3. Proteolytic In-Gel Digestion......Page 221
3.1. Chemical Cross-Linking......Page 222
3.2. SDS-PAGE......Page 223
3.3. In-Gel Digestion......Page 224
3.4. Nano-HPLC/Nano-ESI-LTQ-Orbitrap-MS......Page 225
3.5. Data Analysis......Page 226
4. Notes......Page 229
References......Page 230
1. Introduction......Page 232
2.1. Recombinant Proteins and Inhibitors......Page 233
3. Methods......Page 234
3.1. Protein Sample Preparation: P53/MDM2 Mix......Page 236
3.2. Sample Preparation for High-Mass MALDI Analysis: Control Experiments......Page 237
3.5. Sample Preparation for Inhibition Experiments......Page 238
3.6. Sample Preparation for IC 50 Determination......Page 239
3.7. Data Analysis......Page 240
4. Notes......Page 241
References......Page 242
1. Introduction......Page 244
2.1. Biological Source Material......Page 246
2.4. Generation of Affinity Matrix......Page 247
3.1. Design of Negative Control......Page 248
3.2. Time-Controlled Transcardiac Perfusion Crosslinking......Page 250
3.4. Generation of Affinity Matrix......Page 252
3.5. Affinity-Purification of Bait Protein Complexes......Page 253
3.7. Tandem Mass Spectrometry......Page 254
4. Notes......Page 255
References......Page 258
Part IV: Ligand Discovery\r......Page 260
1. Introduction......Page 262
2.1. Compound Printing and Array Manufacture......Page 264
3. Methods......Page 265
3.1.1. Glass Surface Activation (Fig.  2)......Page 266
3.1.2. Small Molecule Printing (Fig.  3)......Page 267
3.1.3. Catalysis and Quench (Fig.  4)......Page 269
3.2. Screens with Pure Proteins: Detecting Known Interactions with Fkbp12......Page 270
4. Notes......Page 273
References......Page 275
1. Introduction......Page 278
2.2. Kinetic Solubility Measurement......Page 280
3.2. Kinetic Solubility Measurement......Page 282
References......Page 283
1. Introduction......Page 286
3.1. ChEBI Database......Page 287
3.2.5. Chemical Structure Representations......Page 288
3.2.8. Nomenclature......Page 289
3.3. The ChEBI Ontology......Page 290
3.3.2. ChEBI Ontology Structure......Page 291
Simple Search......Page 293
Advanced Text Search......Page 296
Structure Search......Page 297
3.4.2. Browsing......Page 298
getLiteEntity Method......Page 299
getCompleteEntity Method......Page 300
getComplete EntityByList Method......Page 301
getStructureSearch Method......Page 302
Example Workflow for Retrieving the Functional Children of an Entity......Page 303
 OBO File......Page 305
 Database Table Dumps......Page 307
References......Page 308
1. Introduction......Page 310
2.1. Structural Frameworks......Page 311
2.3. Enthalpy and Entropy......Page 313
2.4. Target Resident Time......Page 314
3.2. Number of Aromatic Rings......Page 315
3.4.1. Reactive Functionalities......Page 316
3.4.3. Log P......Page 317
4. Notes......Page 318
References......Page 320
Index\r......Page 322