Glycobiology Methods in Enzymology Volume 415 1st Edition by Minoru Fukuda 0121828204 9780121828202

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ISBN 10: 0121828204

ISBN 13: 9780121828202

Author: Minoru Fukuda

In the past decade, there has been an explosion of progress in understanding the roles of carbohydrates in biological systems. This explosive progress was made with the efforts in determining the roles of carbohydrates in immunology, neurobiology and many other disciplines, examining each unique system and employing new technology. This volume represents the first of three in the Methods in Enzymology series, including Glycomics (vol. 416) and Functional Glycomics (vol. 417), dedicated to disseminating information on methods in determining the biological roles of carbohydrates. These books are designed to provide an introduction of new methods to a large variety of readers who would like to participate in and contribute to the advancement of glycobiology. The methods covered include structural analysis of carbohydrates, biological and chemical synthesis of carbohydrates, expression and determination of ligands for carbohydrate-binding proteins, gene expression profiling including micro array, and generation of gene knockout mice and their phenotype analyses.

Glycobiology Methods in Enzymology Volume 415 1st Table of contents:

Section I: N-Glycan Processing
Chapter 1: Non-Radioactive Analysis of Lipid-Linked Oligosaccharide Compositions by Fluorophore-Assi
Overview
Materials
Procedures
Acknowledgments
References

Chapter 2: Identification of N-Glycan-Binding Proteins for E3 Ubiquitin Ligases
Overview
Binding Protein Assay for N-Glycans
In Vitro Ubiquitination Assay for N-linked Glycoproteins with SCFFbx2
Overlay Assay for the Detection of Fbx2 Binding Proteins
Pull-Down Assay for the Interaction Between Fbx2 and N-Linked Glycoproteins
Interaction of Fbx2 with Integrin ß1 in Neuro2a Cells
Acknowledgments
References

Chapter 3: Family 47 alpha-Mannosidases in N-Glycan Processing
Overview
Overview of Class 1 (GH47) Expression and Purification
Generation of Class 1 alpha-Mannosidase Site-Directed Mutants
Enzyme Expression and Purification
Oligosaccharide Isolation for Enzyme Assays and Binding Studies
alpha-Mannosidase Assays and Kinetic Analyses
Glycan Binding Studies by SPR
Summary
Acknowledgment
References

Chapter 4: A Cytoplasmic Peptide: N-Glycanase
Introduction
In Vitro PNGase Assay
Glycoprotein Substrate for In Vivo PNGase Assay
Technical Considerations
Acknowledgments
References

Section II: Structural Analysis
Chapter 5: Glycomic Profiling of Cells and Tissues by Mass Spectrometry: Fingerprinting and Sequenci
Overview
Preparation of Glycans for Mass Spectrometric Analysis
Enzymatic and Chemical Degradations
Derivatization and Purification of Derivatized Glycans
MS Analyses
Automated Analysis of N-Glycan MALDI Fingerprints
References

Chapter 6: Structural Analysis of Sialyl N-Glycan Using Pyridylamination and Chromatography Followed
Overview
PA-Oligosaccharide Preparation
3-DM Using HPLC
Determination of Structure Using Databases and Partial Degradation
Structural Assignment of MSn Spectral Matching
Acknowledgments
References

Chapter 7: Determination of Glycosylation Sites and Disulfide Bond Structures Using LC/ESI-MS/MS Ana
Overview
Materials
Methods
Identifying Cys-Containing Peptides and N-Linked Glycopeptides
Identifying Disulfide-Bonded Pairs of Peptides
Alkylation of Free Cys Residues (Protocol 1)
Peptide: N-Glycosidase (PNGase) F Digestion (Protocol 2)
Digestion with Trypsin, Chymotrypsin, or Endoproteinase Glu-C (Protocol 3)
Identification of Free Cys-Containing and Disulfide-Bonded Peptides by LC/ESI-MS/MS (Protocol 4)
Complete Reduction with DTT and Alkylation with IAM (Protocol 5)
Notes
Acknowledgments
References

Chapter 8: Identification of O-GlcNAc Sites on Proteins
Introduction
Immunoblot Detection of O-GlcNAc
Site Mapping by Galactosyltransferase Labeling and Edman Degradation
Autogalactosylation of Galactosyltransferase
Galactosyltransferase Labeling
Control Experiments for Galactosyltransferase Labeling
Edman Degradation
Site Mapping by beta-Elimination and Michael Addition with Dithiothreitol
Chemoenzymatic Enrichment of O-GlcNAc-Modified Proteins
Future Directions
Acknowledgments
References

Section III: Carbohydrate Synthesis and Antibiotics
Chapter 9: Chemoenzymatic Synthesis of Glycan Libraries
Introduction
Materials
Methods
Chemical Synthesis of Functionalized Acceptor Substrates
General Isolation and Purification of Synthesized Glycans
General Enzymatic Glycosylation
Other Glycans
References

Chapter 10: Glycoconjugate Vaccines Against Haemophilus influenzae Type b
Overview
Experimental Procedures
References

Chapter 11: Assay of Human Gastric Mucin as a Natural Antibiotic Against Helicobacter pylori
Overview
Culture of H. pylori with AGS Cells Stably Expressing alpha4GlcNAc-Capping Structure
Expression of CD43 Expressing alpha4GlcNAc
Preparation of Human Gastric Mucins
Bacterial Growth Assay
Cell Motility and Morphology
Detection of alpha-D-glucosyl Cholesterol by Mass Spectrometry
In Vitro Assay of Cholesterol alpha-Glucosyltransferase from H. pylori
Continuous Spectrophotometric Assay for Cholesterol alpha-Glucosyltransferases
Future Perspective
Acknowledgments
References

Chapter 12: Molecular Contacts Between Antibiotics and the 30S Ribosomal Particle
Overview
Binding of Non-Aminoglycoside Antibiotics to the 30S Particle
Binding of Aminoglycoside Antibiotics to the 30S Ribosomal Particle
Acknowledgment
References

Chapter 13: Mechanism-Based Inhibitors to Probe Transitional States of Glycoside Hydrolases
Overview
Characterization of Vibrio cholerae Neuraminidase by Using a Mechanism-Based Fluorescent Labeling Re
Synthesis of Suicide Substrate of Neuraminidase 1
Inhibition and Specific Labeling of VCNA
Mechanism-Based Fluorescent Labeling of Neuraminidase
Purification of the Fluorescence-Labeled Peptide by Anti-Dansyl Antibody Column
MALDI-TOF MS and MS/MS Finger Printing Analysis of the Labeled Peptide
References

Chapter 14: Regulating Cell Surface Glycosylation with a Small-Molecule Switch
Overview
Experimental Design
Identification of Localization and Catalytic Domains
Construction of Expression Vectors
Activity Assays of Enzyme Reconstitution
Variables Affecting Enzyme Activity
Location of Catalytic Domains
Applications and Future Directions
References

Chapter 15: Metabolic Labeling of Glycans with Azido Sugars for Visualization and Glycoproteomics
Introduction
Materials and Methods
Synthetic Methods
Metabolic Labeling of Glycans in Cell Culture
Metabolic Labeling of Glycans in Mice
Reaction of Azido Sugars with Labeling Reagents in Cell Culture and in Tissue Lysates
Administration of Labeling Reagents In Vivo
Enrichment and Identification of Metabolically Labeled Glycans
Discussion
Detection of Azide-Labeled Glycans
Enrichment and Identification of Azide-Labeled Glycans
Conclusion
References

Section IV: Carbohydrate Ligand Specificity
Chapter 16: Functional Proteomic Profiling of Glycan-Processing Enzymes
Introduction
General Considerations in Developing Activity-Based Probes
Specific Case: Carbohydrate Processing Enzymes
Case Study: Probing Exoglycosidases in Cell Lysates
Conclusion
Experimental Procedures for Labeling E. coli Cell Lysates Using Probe 6
References

Chapter 17: Oligosaccharide Microarrays to Map Interactions of Carbohydrates in Biological Systems
Introduction
Oligosaccharide Microarrays as Tools in HIV Glycobiology
Microarrays of Synthetic Heparin Oligosaccharides for High-Throughput Screening of Heparin-Protein I
Microarrays of Aminoglycosides
Monosaccharide Microarrays to Screen Interactions of Bacteria and Carbohydrates
Conclusions
Acknowledgments
References

Chapter 18: Identification of Ligand Specificities for Glycan-Binding Proteins Using Glycan Arrays
Overview
Construction of the CFG Streptavidin-Biotin Glycan Array (Plate Glycan Array)
Construction of the CFG Covalent Printed Glycan Array (Printed Glycan Array)
Alternative Assay Conditions
Other Detection Methods and Considerations
Example Data Output From the Plate and Printed Glycan Arrays
References

Chapter 19: High-Throughput Analysis of Lectin-Oligosaccharide Interactions by Automated Frontal Aff
Introduction
Principle of FAC
Experimental
Concluding Remarks
Acknowledgments
References

Chapter 20: Preparation of Neoglycolipids with Ring-Closed Cores via Chemoselective Oxime-Ligation f
Overview
Preparation of the Aminooxy-Functionalized Lipid AOPE
Preparation of Oxime-Linked NGLs (AO-NGLs)
Purification of AO-NGLs
Analysis and Quantitation of Purified AO-NGLs
Status of the Core Monosaccharide in AO-NGLs
Evaluation of AO-NGL s in Carbohydrate-Protein Interaction Studies
Perspectives
References

Chapter 21: Development of a Lectin Microarray Based on an Evanescent-Field Fluorescence Principle
Introduction
Experimental Methods
Results
Concluding Remarks
Acknowledgments
References

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