Glycomics

Series Editors
......Page 1
Copyright
......Page 2
Contributors
......Page 3
Preface
......Page 12
Volume in Series
......Page 13
Mass Spectrometric Analysis of Sulfated N- and O-Glycans......Page 41
Introduction and Overview......Page 42
From biological sources to glycoprotein extracts......Page 45
From glycoproteins to released N- and O-glycans......Page 47
Permethylation and microscale fractionation......Page 48
Methods......Page 49
MS Analyses and Data Interpretation......Page 51
MALDI-based MS and MS/MS analysis......Page 52
Methods......Page 53
Interpretation of MALDI-MS profile of permethylated sulfated glycans......Page 55
CID MS/MS of permethylated sulfated glycans......Page 58
Future Perspectives......Page 61
References......Page 62
Mass Spectrometric Analysis of Mutant Mice......Page 65
Overview......Page 66
Protocol 1: Preparation of samples for glycan release......Page 69
Protocol 2: Preparation of glycans for analysis......Page 72
Protocol 3: Optional sample preparation steps......Page 78
Protocol 4: Derivatization and analysis of released samples......Page 87
Protocol 5: Glycobioinformatics......Page 95
Cartoonist......Page 96
Interpretation of Glycomic Data......Page 97
MALDI-TOF/TOF MS sequencing......Page 100
Enzymatic digestion-α-galactosidase treatment......Page 103
Linkage analysis by GC-MS......Page 105
Summary of Glycan Structural Observations in Murine Tissues, Cells, and Knockouts......Page 107
Acknowledgments......Page 111
References......Page 112
Glycosaminoglycan Characterization by Electrospray Ionization Mass Spectrometry Including Fourier Transform Mass S......Page 116
Overview......Page 118
Preparation of Crude PG/GAG......Page 120
Materials and solutions for CS/DS disaccharide preparation......Page 121
Materials for CS/DS disaccharide analysis......Page 122
Solutions for CS/DS disaccharide analysis......Page 123
Method for CS/DS disaccharide analysis......Page 124
Materials and solutions for HS disaccharide preparation......Page 125
Method for HS disaccharide preparation......Page 126
Materials for HS disaccharide analysis......Page 127
Method for HS disaccharide analysis......Page 128
Direct Infusion ESI FTMS Analysis of Oligosaccharides and Polysaccharides Separated by Preparative Continuous-Elution PAGE.......Page 129
Materials and solutions for GAG release by beta-elimination under reducing conditions......Page 130
Method for GAG release by beta-elimination under reducing conditions......Page 131
Materials and solutions for preparative CE PAGE separation of the bikunin GAG chains......Page 132
Method for preparative CE PAGE separation of the bikunin GAG chains......Page 133
Method for purification of gel-eluted GAG fractions for FTMS analysis......Page 134
Method for the ESI FTMS analysis of gel-eluted bikunin GAG fractions......Page 136
An Approach to FTMS Data Interpretation......Page 137
Method for the ESI FT-ICR MS analysis of bikunin GAG mixture......Page 138
Structural Characterization of GAG Oligosaccharides by Tandem Mass Spectrometry......Page 139
Method for structural characterization of GAG oligosaccharides by tandem MS......Page 140
Summary......Page 142
References......Page 143
Large-Scale Glycomics for Discovering Cancer-Associated N-Glycans by Integrating Glycoblotting and Mass Spectromet......Page 146
Introduction......Page 147
Overview of Glycoblotting Utilized by BlotGlyco Beads......Page 148
Pretreatment and release of N-glycans......Page 149
Enrichment of glycans onto BlotGlyco beads (glycoblotting)......Page 150
Recovery of oligosaccharides from the beads......Page 152
Discovery of glycan-related cancer biomarker utilized by glycoblotting......Page 153
Monitoring mouse ES cell differentiation by N-glycomics utilized by glycoblotting......Page 158
Potential applications for O-glycomics and glycosphingolipidomics......Page 159
The concept of reverse glycoblotting and GFRG......Page 160
References......Page 161
In Vitro and In Vivo Enzymatic Syntheses and Mass Spectrometric Database for N-Glycans and O-Glycans......Page 163
Overview......Page 164
In Vitro Syntheses of N- and O-Linked Glycan Libraries Using Recombinant Human Glycosyltransferases......Page 165
Materials and equipment......Page 166
Preparation of recombinant human enzymes......Page 167
Preparation and purification of PA-labeled N-linked glycan library......Page 169
Construction of O-linked glycopeptide library......Page 170
Conversion of O-linked glycopeptide library to O-linked glycan library......Page 171
Materials and equipment......Page 172
Yeast transformation......Page 173
Expression and purification of MUC1a-6xHis peptide......Page 175
An Application of the Library to Glycan Analysis......Page 177
Construction of an observed tandem mass spectral database......Page 178
How to use the GMDB......Page 179
Navigator System for Glycan Analysis Using the Glycan Mass Spectral Database......Page 182
References......Page 184
Identification of Fucosylated Haptoglobin as a Novel Tumor Marker for Pancreatic Cancer and Its Possible Applicati......Page 186
Introduction......Page 187
Discovery of Fucosylated Haptoglobin as a Marker for Pancreatic Cancer......Page 188
Mass Spectrometry Analysis of the Oligosaccharide Structure on Haptoglobin......Page 189
Identification of an Inducible Factor that Stimulates the Production of Haptoglobin......Page 190
Clinical Application of Lectin-Antibody ELISA to Measure Fucosylated Haptoglobin......Page 193
Acknowledgments......Page 195
References......Page 196
Differential Glycan Profiling by Lectin Microarray Targeting Tissue Specimens......Page 198
Overview......Page 199
Methods for preparation of Cy3-labeled glycoprotein from formalin-fixed tissue sections......Page 201
Methods for lectin microarray analysis and data processing......Page 203
Methods for selection of the best lectin probe......Page 205
Method for dissecting specific regions in tissue sections......Page 207
Differential glycan analysis of a fixed target glycoprotein in tissue sections by antibody-assisted lectin profiling.........Page 209
References......Page 210
A Versatile Technology for Cellular Glycomics Using Lectin Microarray......Page 213
Strategy for Systematic Development of Cell Discrimination Procedures Using Lectin Microarray......Page 214
Production of the Lectin Microarray......Page 215
Data Normalization......Page 217
Glycan Profiles of CHO, Lec2, Lec8, and Lec1......Page 218
Unsupervised Clustering and Principal Component Analysis......Page 221
Significance Difference Test......Page 222
Discriminant Analysis......Page 223
Differential Analysis Between CHO and Lec1......Page 224
Concluding Remarks......Page 225
References......Page 227
Applications of Heparin and Heparan Sulfate Microarrays......Page 228
Introduction......Page 229
Preparation of Amino-Functionalized HS/Heparin Oligosaccharides......Page 232
Microarray Analysis of HS/Heparin-FGF Binding......Page 233
Fabrication of HS/heparin microarrays......Page 235
Binding affinities of HS/heparin with FGFs......Page 237
Materials and equipment......Page 238
Preparation of glycodendrimers and amine-functionalized 5kDa heparin......Page 240
Incubation with FGF-binding heparin oligosaccharide dendrimers......Page 241
Materials and equipment......Page 242
Binding affinities of HS/heparin with chemokines......Page 243
Materials and equipment......Page 244
Conclusions......Page 245
References......Page 246
Measurement of Glycan-Based Interactions by Frontal Affinity Chromatography and Surface Plasmon Resonance......Page 250
Introduction......Page 251
FAC principle......Page 252
Preparation of affinity adsorbents......Page 255
Operation of frontal affinity chromatography......Page 256
Interaction between polySia and neurotransmitter......Page 257
Materials and equipment......Page 258
Immobilization of biotinylated glycans on an Au sensor chip......Page 259
Biacore analysis......Page 260
Conclusions......Page 261
References......Page 262
Detection of Weak-Binding Sugar Activity Using Membrane-Based Carbohydrates......Page 264
Materials......Page 265
Methods......Page 266
Methods......Page 267
Methods......Page 268
Methods......Page 269
Methods......Page 270
References......Page 271
Fluorescence-Based Solid-Phase Assays to Study Glycan-Binding Protein Interactions with Glycoconjugates......Page 272
Overview......Page 273
Biotinylation of Glycopeptides, Oligosaccharides, and Cells......Page 275
Biotinylation of oligosaccharides......Page 276
Labeling of Gal-1 through primary amines......Page 278
Labeling of human T lymphocytes......Page 279
An assay with recombinant P- and L-selectin......Page 280
Determination of apparent binding affinity for L-selectin......Page 281
An assay with T lymphocytes......Page 283
Galectin-1 Binding to Immobilized Glycopeptides and Glycans and Determination of Apparent Binding Affinity......Page 285
An assay with recombinant galectin-1......Page 286
An assay with recombinant galectin-1......Page 288
Determination of apparent binding affinity......Page 290
References......Page 293
Multifaceted Approaches Including Neoglycolipid Oligosaccharide Microarrays to Ligand Discovery for Malectin......Page 296
Overview......Page 297
Materials and equipment......Page 299
Generation of plasmids containing the His6-tagged malectin globular domain......Page 300
Expression and purification of His6-tagged malectin for microarray analysis......Page 301
Materials and equipment......Page 302
Glucan oligosaccharides di- to heptasaccharides......Page 303
Materials and equipment......Page 305
Release and purification of triglucosylated N-glycans from glycoproteins......Page 306
Materials and equipment......Page 307
Isolation of α-glucosidase I......Page 308
Preparation of NGL Probes......Page 309
Preparation of AO-NGLs of the glucosylated N-glycans......Page 310
Materials and equipment......Page 312
Microarray printing......Page 313
Probing the microarrays......Page 314
References......Page 315
Imaging Mass Spectrometry of Glycolipids......Page 318
Overview......Page 319
Methods for preparing the cryosections......Page 320
Matrix Selection and Applying Matrix......Page 321
Methods for applying matrix......Page 322
Measurements by Imaging Mass Spectrometry......Page 323
Data Analyses......Page 324
Identification of Molecules......Page 325
Application of IMS......Page 327
Methods for TLC-Blot-MALDI-IMS......Page 328
References......Page 330
Dynamics and Interactions of Glycoconjugates Probed by Stable-Isotope-Assisted NMR Spectroscopy......Page 333
Assignments of NMR Signals Derived from Glycoprotein Glycan......Page 334
Preparation of selectively labeled IgG-Fc for NMR study......Page 335
Dynamics of Glycoprotein Glycans......Page 337
Determination of amide exchange rates from saturation-transfer experiments......Page 340
Uniform 13C/15N-labeling for NMR assignments of the polypeptide backbone of glycoproteins......Page 341
NMR-Based Screening of Glycopeptides Reactive with Lectin......Page 343
Preparation of glycopeptides for NMR study......Page 344
Perspective......Page 346
References......Page 347
Self and Nonself Recognition with Bacterial and Animal Glycans, Surveys by Synthetic Chemistry......Page 351
Overview......Page 352
Bacterial Glycoconjugates for Nonself Recognition-Lipopolysaccharide (LPS)......Page 353
Glycosylation with Kdo Donor 11......Page 355
Cytokine (IL-6) Induction in Human Peripheral Whole-Blood Cell Cultures......Page 356
Bacterial Glycoconjugates for Nonself Recognition-Peptidoglycan (PGN)......Page 357
Synthesis of Disaccharide Moiety 15 in Tracheal Cytotoxin with beta-Selective Glycosylation......Page 359
Synthesis of Tracheal Cytotoxin 20 and Its Fragment 21......Page 360
Visualizing the In Vivo Dynamics of Animal N-Glycans......Page 361
PET Imaging of Glycoproteins......Page 362
Method for 68Ga-DOTA Labeling and MicroPET Imaging in Rabbit......Page 363
PET Imaging of Glycoclusters......Page 365
Method for Preparation of N-Glycan Clusters and PET Imaging in Mouse......Page 366
Acknowledgments......Page 367
References......Page 368
Multivalent Ligands for Siglecs......Page 371
Sialic acid-binding immunoglobulin-like lectins (Siglecs)......Page 372
Glycan-binding specificity and cell-type expression......Page 375
Cis- and trans-ligand binding......Page 376
Multivalent scaffolds for siglec ligands......Page 377
Reagents and cells......Page 378
Preparation of siglec-expressing cells......Page 379
PAA probe to siglec-expressing cells......Page 380
PAA probes to siglec-Fc beads......Page 383
Siglec-Fc to PAA probe beads......Page 384
Siglec-Fc to biotinylated free saccharide-coated beads......Page 386
Conclusions and Future Directions......Page 387
Acknowledgments......Page 388
References......Page 389
Introduction......Page 392
O-Linked Glycoproteins......Page 394
N-Linked Glycoproteins......Page 401
References......Page 409
Methods to Study the Biosynthesis of Bacterial Furanosides......Page 416
Introduction......Page 417
Discussion......Page 420
Immobilization of GalPUT......Page 421
Generation of UDP-galactofuranose (2)......Page 422
Discussion......Page 425
Materials and methods......Page 426
Kinetic assay......Page 427
Discussion......Page 429
Materials and methods......Page 430
General assay procedure......Page 431
Measuring GlfT2 activity......Page 432
Donor inhibitor screen......Page 433
References......Page 434
The Synthesis of 1,2-cis-Amino Containing Oligosaccharides Toward Biological Investigation......Page 439
References......Page 457
Aminoglycosides: Redesign Strategies
for Improved Antibiotics and
Compounds for Treatment of Human
Genetic Diseases......Page 462
Introduction......Page 463
Aminoglycoside Antibiotics: Their Mode of Action and Major Drawbacks......Page 465
Strategies Toward Development of Improved Antibiotics......Page 468
Alteration of neomycin B at C5 position to prevent APH(3) resistance......Page 469
Dual activity of C5-modified neomycin B derivatives against Bacillus anthracis......Page 472
3,4-Methylidene protected aminoglycosides: A strategy to reduce toxicity and overcome resistance enzymes......Page 474
Neomycin B-based hybrid antibiotics: A strategy to delay resistance development......Page 475
Aminoglycosides as Readthrough Inducers for the Treatment of Genetic Diseases......Page 479
Development of new variants of aminoglycosides with improved readthrough activity and reduced toxicity......Page 480
Acknowledgments......Page 483
References......Page 484
Solid Phase Synthesis of Oligosaccharides......Page 488
Introduction......Page 489
Solid Phase Techniques and Strategies......Page 490
Solid Phase Synthesis of Carbohydrates......Page 493
Automated Solid Phase Synthesis of Carbohydrates......Page 497
Conclusions and Outlook......Page 501
Experimental Data for the Synthesis of Protected Globo-H 47......Page 504
References......Page 506
Novel Synthesis of Functional Mucin Glycopeptides Containing Both N- and O-Glycans......Page 510
Overview......Page 511
Design of MUC1-Related Neoglycoprotein......Page 512
Solid-Phase Glycopeptide Synthesis Using Microwave-Assisted Protocol......Page 513
Procedures......Page 514
Production and Purification of SrtA......Page 515
SrtA-Mediated Ligation......Page 516
Procedures for 3 (Fig.23.2)......Page 517
Procedures for 12 (Fig.23.5)......Page 518
Materials and equipments......Page 519
Procedures for analytical scale for 5 (Fig.23.3)......Page 520
One-Pot Enzymatic Sugar Elongation Catalyzed by Glycosyltransferases......Page 521
Materials and equipments......Page 522
Procedures for 11 (Fig.23.5)......Page 523
BLase-Catalyzed Cleavage from Supporting Polymer......Page 524
Characterization data......Page 525
References......Page 526
Synthesis of Glycopeptides......Page 528
Preparation of N-Linked Complex Type Oligosaccharides......Page 529
Synthesis of asparagines-linked sialyloligosaccharide 2......Page 531
Synthesis of dibenzyl-sialyloligosaccharide 4......Page 533
NDTNTN(sialyloligosaccharide)SSS 5......Page 534
Solid-phase synthesis of sialylglycopeptide-thioester 9......Page 535
Native Chemical Ligation of Glycopeptide......Page 536
Expanding Scope of Native Chemical Ligation......Page 538
Synthesis of 17 (CNBr conversion reaction of sialylglycopeptide 16)......Page 541
References......Page 542
Renewed Synthetic Approach to Gangliosides Exploiting Versatile and Powerful Synthetic Units......Page 545
Background......Page 546
Design of a Systematic Strategy Toward Gangliosides Found in Mammals......Page 547
Renewal of the Synthesis of Sialyl Galactose Unit......Page 549
Sialyl Galactose Unit for Ganglio-Series Ganglioside Synthesis......Page 552
Cassette Approach to Glycolipid Synthesis......Page 553
Synthesis of Echinoderm Ganglioside......Page 555
Materials......Page 558
Note......Page 559
Special precautions......Page 560
Special precautions......Page 561
Materials......Page 562
References......Page 563
Abstract......Page 565
Introduction......Page 566
Ac4-ManNDAz (6)......Page 569
Ac4-GlcNDAz......Page 571
Metabolic Incorporation of Photocrosslinkers into Glycans in Cell Culture......Page 572
Analysis of CMP-Sialic Acids in Cell Lysates by HPAEC......Page 574
Cell Surface Display of SiaDAz in Sialic Acid-Deficient Cells......Page 576
Detection of α2-3 and α2-6 cell surface sialic acid......Page 578
Characterization of SiaDAz-Containing Gangliosides......Page 579
Covalent Capture of Glycan-Mediated Interactions Using Photocrosslinking Sialic Acid......Page 581
Acknowledgments......Page 583
References......Page 584
Identification of Carbohydrate-Binding Proteins by Carbohydrate Mimicry Peptides......Page 587
Materials......Page 588
Visualization of Carbohydrate-Binding Proteins......Page 589
Procedures for peptide affinity chromatography......Page 591
Carbohydrate-binding activity of identified proteins......Page 593
Acknowledgments......Page 594
References......Page 595
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