GLYCOBIOLOGY

Front......Page 1
Table of Contents......Page 2
Contributors to Volume 415......Page 5
Preface......Page 9
Previous Volumes in Methods in Enzymology......Page 10
Analysis of LLO Biosynthesis by Incorporation of Radiolabeled Precursors......Page 34
Approaches for Analysis of Non-Radioactive LLOs......Page 35
LLO Analysis by Fluorophore-Assisted Carbohydrate Electrophoresis......Page 37
Monosaccharide Profiling by FACE......Page 42
LLO and Monosaccharide-P-Dolichol Extraction......Page 43
G0-3M1-9Gn2-P-P-Dol and Free Glycan Analysis: Derivatization with ANTS or ANDS......Page 44
Partial Purification and Separation of Phosphosugars and Nucleotide Sugars......Page 45
Monosaccharide Profiling FACE......Page 46
LLO Extraction and Partial Purification......Page 47
Oligosaccharide Profiling by FACE......Page 49
References......Page 50
[2] Identification of N-Glycan-Binding Proteins for E3 Ubiquitin Ligases......Page 51
Overview......Page 52
Methods......Page 53
Methods......Page 54
Methods......Page 56
Methods......Page 57
Methods......Page 58
References......Page 60
Overview......Page 62
Overview of Class 1 (GH47) Expression and Purification......Page 66
Enzyme Expression and Purification......Page 67
alpha-Mannosidase Assays and Kinetic Analyses......Page 69
Glycan Binding Studies by SPR......Page 71
Summary......Page 73
References......Page 75
Introduction......Page 77
In Vitro PNGase Assay......Page 78
Glycoprotein Substrate for In Vivo PNGase Assay......Page 80
Cell Extracts and Western Blotting......Page 81
Cycloheximide-Decay Experiment......Page 82
Technical Considerations......Page 83
References......Page 84
Overview......Page 87
Materials for Tissue Homogenization......Page 88
Method for Homogenization......Page 89
Method for Tryptic Digestion......Page 90
Method for Release of N-Glycans......Page 91
Method for Release of O-Glycans: Reductive Elimination......Page 92
Method for Desalting of O-Glycans......Page 93
Buffers for Enzymatic Digestions......Page 94
Method for Acid Hydrolysis......Page 95
Method for Permethylation......Page 96
MS Analyses......Page 97
Materials for MALDI Analysis......Page 98
Method for MALDI Analysis......Page 99
Method for Electrospray Ionization Mass Spectrometry (ES-MS)......Page 100
Method for the Preparation of Partially Methylated Alditol Acetates......Page 101
Automated Analysis of N-Glycan MALDI Fingerprints......Page 102
Materials......Page 103
Methods......Page 104
Creating MSD and MSA Files......Page 105
The PARC Mass Spectrum Viewer......Page 107
Menus......Page 111
References......Page 112
Overview......Page 115
Materials and Reagents......Page 116
PA-Oligosaccharide Preparation from Glycoprotein......Page 117
3-DM Using HPLC......Page 118
HPLC Analysis......Page 119
Materials and Methods......Page 122
Structural Assignment of MSn Spectral Matching......Page 125
MS Analysis......Page 126
References......Page 129
[7] Determination of Glycosylation Sites and Disulfide Bond Structures Using LC/ESI-MS/MS Analysis......Page 131
Overview......Page 132
Methods......Page 133
Identifying Cys-Containing Peptides and N-Linked Glycopeptides......Page 134
Identifying Disulfide-Bonded Pairs of Peptides......Page 135
Digestion with Trypsin, Chymotrypsin, or Endoproteinase Glu-C (Protocol 3)......Page 138
Complete Reduction with DTT and Alkylation with IAM (Protocol 5)......Page 139
References......Page 140
[8] Identification of O-GlcNAc Sites on Proteins......Page 141
Introduction......Page 142
Materials......Page 143
Procedure......Page 144
Materials......Page 145
Galactosyltransferase Labeling......Page 146
Procedure......Page 147
Removing O-GlcNAc by Hexosaminidase Digestion......Page 149
Procedure for Coupling the Sample the PVDF Disk for Manual Edman Degradation......Page 150
Site Mapping by beta-Elimination and Michael Addition with Dithiothreitol......Page 151
Materials......Page 152
Protein Digestion......Page 153
Mild BEMAD Treatment......Page 154
Thiol Affinity Chromatography......Page 155
Chemoenzymatic Enrichment of O-GlcNAc-Modified Proteins......Page 156
Procedure......Page 157
Future Directions......Page 158
References......Page 159
Introduction......Page 162
Enzyme Production Materials......Page 163
General Procedure for the Bacterial Expression......Page 164
General Procedure for the Mammalian Expression System......Page 166
Enzyme Purification......Page 167
Dowex Resin and Size Exclusion Chromatography......Page 168
Preparative High-Performance Liquid Chromatography......Page 169
Notes......Page 170
Notes......Page 172
O-Glycans......Page 173
Globoside Glycans......Page 174
References......Page 175
Overview......Page 178
General Methods......Page 183
Conjugation to Tetanus Toxoid......Page 185
References......Page 187
Overview......Page 189
Culture of H. pylori with AGS Cells Stably Expressing alpha4GlcNAc-Capping Structure......Page 191
Expression of CD43 Expressing alpha4GlcNAc......Page 192
Preparation of Human Gastric Mucins......Page 194
Cell Motility and Morphology......Page 195
Detection of alpha-D-glucosyl Cholesterol by Mass Spectrometry......Page 196
In Vitro Assay of Cholesterol alpha-Glucosyltransferase from H. pylori......Page 197
Continuous Spectrophotometric Assay for Cholesterol alpha-Glucosyltransferases......Page 198
Future Perspective......Page 201
References......Page 202
Overview......Page 205
Tetracycline......Page 209
Spectinomycin......Page 210
Pactamycin......Page 212
Hygromycin......Page 214
Streptomycin......Page 216
Neamine-Containing Aminoglycosides and Apramycin......Page 218
Binding of Neamine-Containing Aminoglycosides to Mutant Ribosomes......Page 220
Acknowledgment......Page 223
References......Page 224
Overview......Page 227
Characterization of Vibrio cholerae Neuraminidase by Using a Mechanism-Based Fluorescent Labeling Reagent......Page 229
Synthesis of Suicide Substrate of Neuraminidase 1......Page 230
Inhibition and Specific Labeling of VCNA......Page 231
Purification of the Fluorescence-Labeled Peptide by Anti-Dansyl Antibody Column......Page 232
MALDI-TOF MS and MS/MS Finger Printing Analysis of the Labeled Peptide......Page 233
References......Page 236
Overview......Page 238
Experimental Design......Page 239
Identification of Localization and Catalytic Domains......Page 241
Localization Domain Plasmids......Page 242
Catalytic Domain Plasmids......Page 243
Flow Cytometry Analysis of Cell Surface Glycoproteins......Page 244
Day 3: Split Cells and Add Rapamycin......Page 245
Western Blot Analysis of Secreted Glycoproteins......Page 246
Variables Affecting Enzyme Activity......Page 247
Immunofluorescence Microscopy......Page 251
Applications and Future Directions......Page 252
References......Page 253
Introduction......Page 255
Materials and Methods......Page 258
Ac4ManNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetyl-alpha,beta-D-mannosamine......Page 259
Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetyl-alpha,beta-D-galactosamine......Page 260
Phosphine-PFP (2-diphenylphosphanyl-terephthalic acid 1-methyl ester 4-pentafluorophenyl ester)......Page 262
Metabolic Labeling of Glycans in Cell Culture......Page 263
Reaction of Azido Sugars with Labeling Reagents in Cell Culture and in Tissue Lysates......Page 264
Enrichment and Identification of Metabolically Labeled Glycans......Page 266
Discussion......Page 267
Detection of Azide-Labeled Glycans......Page 269
Enrichment and Identification of Azide-Labeled Glycans......Page 270
Further Reading......Page 273
Introduction......Page 276
General Considerations in Developing Activity-Based Probes......Page 277
Specific Case: Carbohydrate Processing Enzymes......Page 279
Case Study: Probing Exoglycosidases in Cell Lysates......Page 282
Step 2: Chemoselective Ligation Reaction......Page 286
Step 3: Gel-Base Profiling of the Proteome......Page 287
Experimental Procedures for Labeling E. coli Cell Lysates Using Probe 6......Page 288
References......Page 290
Introduction......Page 292
Oligosaccharide Microarrays as Tools in HIV Glycobiology......Page 293
Materials and Equipment......Page 294
Preparation of High-Mannose Microarrays......Page 295
Hybridization Experiments......Page 296
Microarrays of Synthetic Heparin Oligosaccharides for High-Throughput Screening of Heparin-Protein Interactions......Page 297
Preparation of Heparin Arrays......Page 300
Incubation with Heparin-Binding Proteins......Page 301
Microarrays of Aminoglycosides......Page 302
Materials and Equipment......Page 303
Preparation of Aminoglycoside-Bearing Microarrays......Page 304
Synthesis of RNA for Microarray Applications......Page 306
Hybridization of RNA to Aminoglycoside Microarrays......Page 307
Hybridization of Proteins to Aminoglycoside Microarrays......Page 308
Materials and Equipment......Page 309
Preparation of Carbohydrate Microarrays......Page 310
Hybridization of Bacteria with Carbohydrate Microarrays......Page 311
References......Page 312
[18] Identification of Ligand Specificities for Glycan-Binding Proteins Using Glycan Arrays......Page 315
Overview......Page 316
Construction of the CFG Streptavidin‐Biotin Glycan Array (Plate Glycan Array)......Page 317
Direct Binding Assays......Page 319
Analysis of Binding Specificity on the CFG Plate Array......Page 320
Construction of the CFG Covalent Printed Glycan Array (Printed Glycan Array)......Page 321
Direct Binding Assays......Page 322
Analyzing the Covalent Printed Array Results......Page 323
Assays of Cells and Organisms......Page 324
Fluorescence Reporters Used in Glycan Array Assays......Page 325
Example Data Output From the Plate and Printed Glycan Arrays......Page 329
References......Page 331
Introduction......Page 334
Principle of FAC......Page 335
Immobilization of Lectins......Page 337
Preparation of Miniature Column......Page 339
Operation of FAC......Page 340
Full Specificity Analysis Using More Than 100 Glycans......Page 342
Concentration Dependence Analysis......Page 345
Concluding Remarks......Page 347
References......Page 348
Overview......Page 349
Materials......Page 351
Step 2: Preparation of N-aminooxyacetyl-DHPE (AOPE)......Page 352
Materials......Page 353
Preparation of NGLs from Other Saccharides......Page 354
Materials......Page 355
Semi-Preparative HPTLC Purification Procedure......Page 356
Status of the Core Monosaccharide in AO-NGLs......Page 357
Preparation of Liposomes......Page 359
Procedures......Page 360
References......Page 362
Introduction......Page 364
Evanescent-Field Fluorescence-Assisted Microarray System......Page 365
Fabrication of Lectin Microarray......Page 366
Scanning and Data Analysis......Page 367
Validation of Specific Binding Between Oligosaccharides and Immobilized Lectins......Page 368
Real-Time Scanning of Binding Reactions......Page 369
Application of Glycoprotein Probes on the Lectin-Antibody Hybrid-Array Slide......Page 370
Concluding Remarks......Page 371
Acknowledgments......Page 372
References......Page 373
Author Index......Page 375
Subject Index......Page 397