A Laboratory Guide to Glycoconjugate Analysis

1 High-Performance Liquid Chromatography of Derivatized and Non-Derivatized Oligosaccharides: A Review.- 1.1 Introduction.- 1.2 Separation Techniques.- Size-exclusion chromatography.- Ion-exchange chromatography.- Hydrophilic-interaction chromatography.- Reversed-phase chromatography.- Mixed elution protocols.- 1.3 Detection Techniques.- Refractometer.- UV detector.- Fluorescence and photometric detection after derivatization.- Detection after Postcolumn Reaction.- Miscellaneous Detection Techniques.- References.- 2 Detection of the Presence of Carbohydrates in Glycoproteins.- 2.1 Introduction.- A general chemical based method for glycoprotein identification.- 2.2 Principle.- Substances interfering with detection.- 2.3 Glycoprotein Detection on Membranes or in Solution: Relative Merits of Protocols.- Membrane labelling.- Solution labelling.- Sensitivity of detection.- Initial experiments.- Interpretation of results and troubleshooting.- Additional analyses: Alternative detection techniques.- 2.4 Identification of Specific Monosaccharides or Glycans.- Lectins.- Monoclonal antibodies to carbohydrate determinants.- Glycosyl transferases.- Naturally occurring carbohydrate recognition proteins.- Glycosyl-phosphatidyl-inositol anchor detection.- 2.5 Detailed Analysis of Protein Glycosylation Release-Label-Profile.- Acknowlegements.- References.- 3 HPAE-PAD Monosaccharide and Oligosaccharide Analysis of Glycoproteins Electrotransferred onto Polyvinylidene Fluoride Membranes.- 3.1 Introduction.- 3.2 SDS-PAGE.- 3.3 Electroblotting and Staining.- 3.4 Monosaccharide Composition Analysis.- Chromatography, detection and quantification of monosaccharides.- 3.5 Oligosaccharide Mapping.- Acknowledgement.- References.- 4 Determination of the Disaccharide Composition of Glycosaminoglycans: Comparison of Chemical and Enzymatic Scission.- 4.1 Introduction.- 4.2 Technical Procedures.- Sample preparation.- 4.3 Troubleshooting.- Enzyme digests.- Chemical depolymerization.- HPLC analysis.- Acknowledgements.- References.- 5 Mass Spectrometric Analysis of Highly Acidic Polysaccharides.- 5.1 Introduction.- 5.2 Principles of Matrix-Assisted Laser Desorption.- 5.3 Technical Procedures.- Sample preparation.- 5.4 Troubleshooting.- References.- 6 Analysis of N-Glycans by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.- 6.1 Introduction.- 6.2 MALDI-MS: Principle of Operation.- Instrumentation.- Analytical protocols.- 6.3 Analysis of Glycoproteins.- Glycoprotein De-N-glycosylation with PNGase F.- MALDI-MS of glycoproteins.- 6.4 Analysis of Glycopeptides.- Structural analysis of N-glycans by MALDI-MS of glycopeptides in combination with exoglycosidase array sequencing.- MALDI-MS of glycopeptides.- 6.5 Analysis of Released Oligosaccharides.- Analysis of released N-glycans by MALDI-MS.- MALDI-MS of glycans.- 6.6 Analysing Carbohydrate Mass Information.- Quantitation.- Accessing the complex carbohydrate structure database.- Data for calculation of oligosaccharide masses.- References.- 7 Polyacrylamide Gel Electrophoresis of Fluorophore-Labelled Reducing Saccharides: A Review.- 7.1 Introduction.- 7.2 Principle.- 7.3 Preparation of Wheat Starch Digest Electrophoretic Standard.- Troubleshooting.- 7.4 Enzymatic Release of Asparagine-Linked Oligosaccharides from Glycoproteins Using PNGase F.- Troubleshooting.- 7.5 Release of Asparagine and Serine/Threonine-Linked Glycans from Glycoproteins Using Hydrazinolysis.- 7.6 Derivatization of the Oligosaccharides with ANTS.- 7.7 Derivatization of Oligosaccharides with AMAC.- 7.8 PAGE for the Separation of ANTS-Derivatized Oligosaccharides and for Acidic Oligosaccharides Derivatized with AMAC.- Troubleshooting.- 7.9 PAGE for the Separation of Neutral and Acidic AMAC-Derivatized Saccharides.- 7.10 Enzymatic Structural Analysis of N-Glycans.- 7.11 Viewing and Imaging the Electrofluorograms.- References.- 8 Carbohydrate Analysis with Capillary Electrophoresis.- 8.1 Introduction.- Instrumentation.- Capillaries.- 8.2 Sample Preparation.- Glycoprotein and oligosaccharide hydrolysis for compositional analysis.- Release of complex oligosaccharides.- Enzymatic cleavage.- Derivatization.- 8.3 Capillary Electrophoretic Separations.- Monosaccharides.- Complex oligosaccharides.- Glycoproteins.- References.- 9 Oligosaccharide Profiling of Keratan Sulphate.- 9.1 Introduction.- 9.2 Technical Procedures.- Keratan sulphate preparation from tissues.- KS molecular weight determination.- Oligosaccharide profiling.- Hydrazinolysis/nitrous acid.- Keratanase (EC 3.2.1.103).- Keratanase II (EC 3.2.1.-from Bacillus sp.).- Chromatography.- Borotritiide reduction and [35S]- labelled samples.- Acknowledgements.- References.- 10 Analysis of the Structure of Heparin and Heparan Sulfate by High-Resolution Separation of Oligosaccharides.- 10.1 Introduction.- 10.2 Technical Procedures.- 10.3 Method Selection, Critical Parameters and Troubleshooting.- Anticipated results.- Time considerations.- References.- 11 HPLC Strategies for Profiling and Sequencing Oligosaccharides.- 11.1 Introduction.- Oligosaccharide profiling.- Detailed structural analysis.- 11.2 HPLC Separations Technology.- Normal phase HPLC.- Choice of column.- Optimization of solvents and gradients.- Profiling and sequencing.- Weak anion-exchange HPLC.- Reversed-phase HPLC.- 11.3 Requirements of HPLC System for Glycan Analysis.- HPLC hardware.- Buffer system15% of another glycan species.- Insufficient sample (of the Technique.- 14.3 Release of Glycans by Hydrazinolysis.- 14.4 Derivatizing the Released Glycans with PA.- 14.5 Analysis and Confirmation of PA-Glycans by 2D CE Using Fluorimetrie Detection.- 14.6 Troubleshooting Guide.- References.- 15 The Application of Three-Dimensional HPLC to the Identification of N-linked Oligosaccharide Structures.- 15.1 Introduction.- 15.2 Isolation and Identification of Neutral and Sialyl PA-Oligosaccharides by Three Successive HPLC Columns.- 15.3 Technical Procedures.- Differentiation between Neu5Ac-?-(2,6)- and Neu5Ac-?-(2/3)-containing oligosaccharides.- Differentiation between Neu5Gc- and Neu5Ac-containing oligosaccharides.- 15.4 Notes.- References.- 16 Simultaneous Fluorescent Labelling and Biotinylation of Oligosaccharides: A Versatile Approach to the Analysis of Oligosaccharide Structure and Function.- 16.1 Introduction.- 16.2 Synthesis and Purification of BAP.- Troubleshooting guide.- 16.3 Coupling of Oligosaccharides to BAP.- Troubleshooting guide.- 16.4 Purification of BAP Oligosaccharides.- Troubleshooting guide.- 16.5 Fractionation and Structural Analysis of BAP Adducts.- Troubleshooting guide.- 16.6 Preparation of BAP-Oligosaccharide Neoglycoconjugates With Streptavidin or Avidin and Their Applications.- Troubleshooting guide.- 16.7 Comparison of BAP With Other Currently Available Fluorescent Tags.- References.- 17 Preparation of Neoglycolipids for Structure and Function Assignments of Oligosaccharides.- 17.1 Introduction.- 17.2 Preparation of Neoglycolipids (Conjugation of Oligosaccharides).- 17.3 Separation and Purification of Neoglycolipids.- 17.4 Visualization and Quantitation of Neoglycolipids.- Densitometry.- 17.5 Chemical Analysis of Neoglycolipids.- Characterization of neoglycolipids by MS.- MS.- 17.6 Enzymatic and Chemical Modifications of Neoglycolipids.- 17.7 Troubleshooting.- References.- 18 Analysis of Protein -1 High-Performance Liquid Chromatography of Derivatized and Non-Derivatized Oligosaccharides: A Review.- 1.1 Introduction.- 1.2 Separation Techniques.- Size-exclusion chromatography.- Ion-exchange chromatography.- Hydrophilic-interaction chromatography.- Reversed-phase chromatography.- Mixed elution protocols.- 1.3 Detection Techniques.- Refractometer.- UV detector.- Fluorescence and photometric detection after derivatization.- Detection after Postcolumn Reaction.- Miscellaneous Detection Techniques.- References.- 2 Detection of the Presence of Carbohydrates in Glycoproteins.- 2.1 Introduction.- A general chemical based method for glycoprotein identification.- 2.2 Principle.- Substances interfering with detection.- 2.3 Glycoprotein Detection on Membranes or in Solution: Relative Merits of Protocols.- Membrane labelling.- Solution labelling.- Sensitivity of detection.- Initial experiments.- Interpretation of results and troubleshooting.- Additional analyses: Alternative detection techniques.- 2.4 Identification of Specific Monosaccharides or Glycans.- Lectins.- Monoclonal antibodies to carbohydrate determinants.- Glycosyl transferases.- Naturally occurring carbohydrate recognition proteins.- Glycosyl-phosphatidyl-inositol anchor detection.- 2.5 Detailed Analysis of Protein Glycosylation Release-Label-Profile.- Acknowlegements.- References.- 3 HPAE-PAD Monosaccharide and Oligosaccharide Analysis of Glycoproteins Electrotransferred onto Polyvinylidene Fluoride Membranes.- 3.1 Introduction.- 3.2 SDS-PAGE.- 3.3 Electroblotting and Staining.- 3.4 Monosaccharide Composition Analysis.- Chromatography, detection and quantification of monosaccharides.- 3.5 Oligosaccharide Mapping.- Acknowledgement.- References.- 4 Determination of the Disaccharide Composition of Glycosaminoglycans: Comparison of Chemical and Enzymatic Scission.- 4.1 Introduction.- 4.2 Technical Procedures.- Sample preparation.- 4.3 Troubleshooting.- Enzyme digests.- Chemical depolymerization.- HPLC analysis.- Acknowledgements.- References.- 5 Mass Spectrometric Analysis of Highly Acidic Polysaccharides.- 5.1 Introduction.- 5.2 Principles of Matrix-Assisted Laser Desorption.- 5.3 Technical Procedures.- Sample preparation.- 5.4 Troubleshooting.- References.- 6 Analysis of N-Glycans by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.- 6.1 Introduction.- 6.2 MALDI-MS: Principle of Operation.- Instrumentation.- Analytical protocols.- 6.3 Analysis of Glycoproteins.- Glycoprotein De-N-glycosylation with PNGase F.- MALDI-MS of glycoproteins.- 6.4 Analysis of Glycopeptides.- Structural analysis of N-glycans by MALDI-MS of glycopeptides in combination with exoglycosidase array sequencing.- MALDI-MS of glycopeptides.- 6.5 Analysis of Released Oligosaccharides.- Analysis of released N-glycans by MALDI-MS.- MALDI-MS of glycans.- 6.6 Analysing Carbohydrate Mass Information.- Quantitation.- Accessing the complex carbohydrate structure database.- Data for calculation of oligosaccharide masses.- References.- 7 Polyacrylamide Gel Electrophoresis of Fluorophore-Labelled Reducing Saccharides: A Review.- 7.1 Introduction.- 7.2 Principle.- 7.3 Preparation of Wheat Starch Digest Electrophoretic Standard.- Troubleshooting.- 7.4 Enzymatic Release of Asparagine-Linked Oligosaccharides from Glycoproteins Using PNGase F.- Troubleshooting.- 7.5 Release of Asparagine and Serine/Threonine-Linked Glycans from Glycoproteins Using Hydrazinolysis.- 7.6 Derivatization of the Oligosaccharides with ANTS.- 7.7 Derivatization of Oligosaccharides with AMAC.- 7.8 PAGE for the Separation of ANTS-Derivatized Oligosaccharides and for Acidic Oligosaccharides Derivatized with AMAC.- Troubleshooting.- 7.9 PAGE for the Separation of Neutral and Acidic AMAC-Derivatized Saccharides.- 7.10 Enzymatic Structural Analysis of N-Glycans.- 7.11 Viewing and Imaging the Electrofluorograms.- References.- 8 Carbohydrate Analysis with Capillary Electrophoresis.- 8.1 Introduction.- Instrumentation.- Capillaries.- 8.2 Sample Preparation.- Glycoprotein and oligosaccharide hydrolysis for compositional analysis.- Release of complex oligosaccharides.- Enzymatic cleavage.- Derivatization.- 8.3 Capillary Electrophoretic Separations.- Monosaccharides.- Complex oligosaccharides.- Glycoproteins.- References.- 9 Oligosaccharide Profiling of Keratan Sulphate.- 9.1 Introduction.- 9.2 Technical Procedures.- Keratan sulphate preparation from tissues.- KS molecular weight determination.- Oligosaccharide profiling.- Hydrazinolysis/nitrous acid.- Keratanase (EC 3.2.1.103).- Keratanase II (EC 3.2.1.-from Bacillus sp.).- Chromatography.- Borotritiide reduction and [35S]- labelled samples.- Acknowledgements.- References.- 10 Analysis of the Structure of Heparin and Heparan Sulfate by High-Resolution Separation of Oligosaccharides.- 10.1 Introduction.- 10.2 Technical Procedures.- 10.3 Method Selection, Critical Parameters and Troubleshooting.- Anticipated results.- Time considerations.- References.- 11 HPLC Strategies for Profiling and Sequencing Oligosaccharides.- 11.1 Introduction.- Oligosaccharide profiling.- Detailed structural analysis.- 11.2 HPLC Separations Technology.- Normal phase HPLC.- Choice of column.- Optimization of solvents and gradients.- Profiling and sequencing.- Weak anion-exchange HPLC.- Reversed-phase HPLC.- 11.3 Requirements of HPLC System for Glycan Analysis.- HPLC hardware.- Buffer system15% of another glycan species.- Insufficient sample (of the Technique.- 14.3 Release of Glycans by Hydrazinolysis.- 14.4 Derivatizing the Released Glycans with PA.- 14.5 Analysis and Confirmation of PA-Glycans by 2D CE Using Fluorimetrie Detection.- 14.6 Troubleshooting Guide.- References.- 15 The Application of Three-Dimensional HPLC to the Identification of N-linked Oligosaccharide Structures.- 15.1 Introduction.- 15.2 Isolation and Identification of Neutral and Sialyl PA-Oligosaccharides by Three Successive HPLC Columns.- 15.3 Technical Procedures.- Differentiation between Neu5Ac-?-(2,6)- and Neu5Ac-?-(2/3)-containing oligosaccharides.- Differentiation between Neu5Gc- and Neu5Ac-containing oligosaccharides.- 15.4 Notes.- References.- 16 Simultaneous Fluorescent Labelling and Biotinylation of Oligosaccharides: A Versatile Approach to the Analysis of Oligosaccharide Structure and Function.- 16.1 Introduction.- 16.2 Synthesis and Purification of BAP.- Troubleshooting guide.- 16.3 Coupling of Oligosaccharides to BAP.- Troubleshooting guide.- 16.4 Purification of BAP Oligosaccharides.- Troubleshooting guide.- 16.5 Fractionation and Structural Analysis of BAP Adducts.- Troubleshooting guide.- 16.6 Preparation of BAP-Oligosaccharide Neoglycoconjugates With Streptavidin or Avidin and Their Applications.- Troubleshooting guide.- 16.7 Comparison of BAP With Other Currently Available Fluorescent Tags.- References.- 17 Preparation of Neoglycolipids for Structure and Function Assignments of Oligosaccharides.- 17.1 Introduction.- 17.2 Preparation of Neoglycolipids (Conjugation of Oligosaccharides).- 17.3 Separation and Purification of Neoglycolipids.- 17.4 Visualization and Quantitation of Neoglycolipids.- Densitometry.- 17.5 Chemical Analysis of Neoglycolipids.- Characterization of neoglycolipids by MS.- MS.- 17.6 Enzymatic and Chemical Modifications of Neoglycolipids.- 17.7 Troubleshooting.- References.- 18 Analysis of Protein - Carbohydrate Interactions by FACE/Gel Retardation Assay.- 18.1 Introduction.- 18.2 Principle of FACE/Gel Retardation Assay.- 18.3 Labelling of Oligosaccharides with ANTS.- 18.4 Screening of Carbohydrate Ligands for Proteins.- 18.5 Measurement of Binding Constant for the Interaction Between Protein and ANTS-Labelled Carbohydrate.- 18.6 Measurement of Binding Constant for the Interaction Between Protein and Native Carbohydrate.- References.- 19 The Application of Capillary Affinity Electrophoresis to the Analysis of Carbohydrate-Protein Interactions.- 19.1 Introduction.- 19.2 Principle of CAE.- 19.3 Determination of Association Constants.- 19.4 Technical Procedures.- General considerations.- 19.5 Limitations of the Technique.- 19.6 Application of CAE to the Analysis of Carbohydrate-Protein Interactions.- 19.7 Conclusions.- References.- 20 Analysis of Protein-Glycosaminoglycan Interactions by Affinity Co-Electrophoresis.- 20.1 Introduction.- Definitions.- 20.2 Technical Procedures.- 20.3 Sample Detection and Sample Recovery.- Autoradiography and staining.- Sample detection by blotting.- Semipreparative ACE.- 20.4 Analysis of Data.- Measuring sample mobilities - calculating a retardation coefficient.- Graphical analysis of data.- Interpreting ACE patterns.- Reverse ACE.- 20.5 Summary.- Acknowledgements.- References.