Main description:

The growing importance of glycobiology and carbohydrate chemistry in modern biotechnology and the pharmaceutical industry makes accurate carbohydrate analysis indispensable. This book provides the principles and protocols of various fundamental carbohydrate analysis methods. Choice of method is entirely dependent upon the type of material being investigated (biological samples, food products, etc.), and the level of structural detail required, i.e. sugar content, compositional analysis, linkages between the sugar components, or the total chemical structure of a given molecule. Full structural characterization of carbohydrate chains requires significant time, resources, and skill in several methods of analysis; no single technique can address all glycan analysis needs.
This book summarizes several existing analytical techniques (both chemical and physical) in an introductory volume designed for the non-expert researcher or novice scientist. While background in carbohydrate chemistry is assumed, all information necessary to understanding the described techniques is addressed in the text.

Contents:

List of abbreviationsPreface

Introduction

Chapter 1 The World of Carbohydrates

Chapter 2 Basic Knowledge of Glycobiology

2.1 Monosaccharides

2.1.1 The Molecular Structure

2.1.2 D/L-forms of Monosaccharides

2.1.3 Specific Monosaccharides

2.2 Oligosaccharides

2.3 Polysaccharides

2.4 Glycoconjugates

2.4.1 Glycoproteins

2.4.1.1 N-glycans

2.4.1.2 O-glycans

2.4.1.3 Blood Group Determinants

2.4.1.4 Different Types of Glycosylation

2.4.1.5 Glycosides

2.4.2 Proteoglycans and Glycosaminoglycans

2.4.3 Glycolipids

2.4.4 Glycosylphosphatidyl-inositol Membrane Anchors

2.5 Intrinsic Complexity of Carbohydrates

2.6 Working in a Laboratory

Chapter 3 Carbohydrates involved in Diseases

Chapter 4 Detection of Carbohydrate by Colorimetric Methods

4.1 Introduction

4.2 Preparing Standard Sugar Solutions for Calibration Curves

4.3 General tests

4.3.1 Detection of Carbohydrate by a Spot Test

4.3.2 Phenol-Sulfuric Acid Assay

4.3.3 Anthrone Test

4.3.4 Molisch's Test

4.3.5 Fehling's Test

4.3.6 Benedict's Test

4.3.7 Barfoed's Test

4.4 Specific Tests

4.4.1 Seliwanoff's Test for Ketoses

4.4.2 Bial's Test for Pentoses

4.4.3 Colorimetric Analysis of (N-acetyl) Aminosugars

4.4.4 Colorimetric Analysis of Uronic Acids

4.4.5 Colorimetric Analysis of Sialic Acids

4.4.6 Iodine Test for Starch

Chapter 5. Analytical Techniques to Study Carbohydrates

5.1 Structural Parameters

5.2 Glycosidic-bond Cleavage techniques

5.2.1 Sulfuric Acid Hydrolysis

5.2.2 Hydrochloric Acid Hydrolysis

5.2.3 Trifluoroacetic Acid Hydrolysis

5.2.4 Methanolysis

5.2.5 Formolysis

5.2.6 Acetolysis

5.2.7 Specific Degradations

5.3 Common Separation Techniques for Mono/Oligosaccharides

5.3.1 Thin-Layer Chromatography

5.3.2 Size-Exclusion Chromatography

5.3.3 High-Performance Liquid Chromatography

5.3.4 Porous Graphitized Carbon Chromatography

5.3.5 Anion/Cation-Exchange Chromatography

5.3.6 High-pH Anion-Exchange Chromatography

5.3.7 Affinity Chromatography

5.4 Detection Techniques for Liquid Chromatography

5.4.1 Detectors

5.4.2 Labeling of Glycans for Detection

5.4.2.1 Labeling by Reductive Amination

5.4.2.2 Labeling by Michael Addition with PMP

5.4.2.3 Permethylation of Glycans for Mass Spectrometry

5.5 Electrophoretic Separation of Carbohydrates

5.6 Gas-Liquid Chromatography

Chapter 6 Monosaccharide Composition Analysis

6.1 Introduction

6.2 Gas-chromatographic Monosaccharide Analysis using TMS Methyl Glycosides

6.3 Gas-chromatographic Monosaccharide Analysis using Alditol Acetates

6.4 Determination of D/L Configuration of Monosaccharides

6.5 Monosaccharide Analysis by HPLC Methods 6.5.1 High-pH Anion-Exchange Chromatography

6.5.2 Ultra-High-Performance Liquid Chromatography

6.6 Determination of Carbohydrate Linkages by Methylation Analysis

6.6.1 Chemical Aspects

6.6.2 GLC-EI Mass Spectrometry of PMAAs

6.6.3 Glycosidic Linkage Determination by HPLC-MS/MS

Chapter 7 Carbohydrate Analysis of Glycoconjugates

7.1 Introduction

7.2 Analysis of Glycoproteins

7.2.1 A Typical Approach to the Analysis of a Glycoprotein

7.2.2 Chemical Release of N- and O-linked Glycans

7.2.3 Enzymatic Release of N-linked Glycans

7.2.3.1 Isolation of N-glycans and de-N-glycosylated

protein (containing O-glycans)

7.2.4 Release of O-glycans by Reductive -elimination

7.3 Analysis of Glycopeptides

7.3.1 Preparation of Glycopeptides from Glycoproteins

7.3.2 Separation of Glycopeptides

7.4 Analysis of Proteoglycans and their Glycosaminoglycans

7.5 Analysis of Glycolipids

7.5.1 General Aspects

7.5.2 Thin-Layer Chromatography of Glycolipids

7.5.3 Carbohydrates of Glyco(sphingo)lipids

7.5.4 Carbohydrates of Glycosylphosphatidylinositol (GPI) Anchors

7.6 Analysis of Polysaccharides

Chapter 8 Structural Characterization of Released Glycans

8.1 Introduction

8.2 Analysis of N-glycans

8.2.1 Fractionation of N-glycans

8.2.2 Preparation of Glucose Oligomers Standard (Dextran-ladder)

8.2.3 Enzymatic Sequence Analysis of N-glycans

8.3 Analysis of O-glycans

8.3.1 General Aspects

8.3.2 Mucin Glycoproteins

8.3.3 Fractionation of O-glycans

Chapter 9 Analysis of Sialic Acids

9.1 General Aspects

9.2 Characterization of Sialic Acid Residues

9.2.1 Colorimetric Determination

9.2.2 Gas-chromatographic Determination

9.2.3 Release of Sialic Acids by Mild Acid Hydrolysis

9.2.4 Analysis of DMB-labeled Sialic Acid

9.2.5 Release of Sialic Acids by Enzymatic Digestion

9.3 Mass Spectrometry of Sialic Acids

9.4 1H NMR Spectroscopy of Sialic Acids

Chapter 10 Carbohydrate Microarray Technology

Chapter 11 Analysis of Carbohydrates by Mass Spectrometry

11.1 General Aspects

11.2 Electron Impact Mass Spectrometry (EI-MS)

11.3 Mass Spectrometry of Glycans

11.3.1 General

11.3.2 Matrix-Assisted Laser Desorption/ionization-Time of Flight mass

spectrometry (MALDI-TOF-MS)

11.3.3 Electrospray Ionization-Mass Spectrometry (ESI-MS)

11.3.4 Tandem Mass Spectrometry

11.3.5 Mass Fragmentation of Oligosaccharides

Chapter 12 Analysis of Carbohydrates by Nuclear Magnetic

Resonance Spectroscopy

12.1 Introduction

12.2 Principle of NMR Spectroscopy

12.3 NMR Spectroscopy of Carbohydrates

12.3.1 Sample Preparation and Measuring

12.3.2 1H NMR Structural-Reporter-Group Concept

12.3.3 13C NMR Spectroscopy

12.4 NMR Spectroscopy for Glycans

12.4.1 Two-dimensional NMR Spectroscopy

12.4.2 Spectral Interpretation and Assignments

12.5 NMR Spectroscopy of Polysaccharides

12.5.1 Summarized NMR Measuring Procedure

Chapter 13 Glycobioinformatics

Chapter 14 Concluding Remarks

Appendix A EI-MS of partially methylated alditol acetates (PMAAs)

Appendix B MALDI-TOF-MS of permethylated N-glycans

Appendix C ES-MS [M-H]- signals of some O-glycans (as alditols)

Glossary of terms

ACKNOWLEDGMENTS

DISCLAIMER

REFERENCES

Subject Index