Main description:

18. 2 Principle of FACE/Gel Retardation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 18. 3 Labelling of Oligosaccharides with ANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 18. 4 Screening of Carbohydrate Ligands for Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 18. 5 Measurement of Binding Constant for the Interaction Between Protein and ANTS-Labelled Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 18. 6 Measurement of Binding Constant for the Interaction Between Protein and Native Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 ~ The Application of Capillary Affinity Electrophoresis to the Analysis _ of Carbohydrate-Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 19. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 19. 2 Principle of CAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 19. 3 Determination of Association Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 19. 4 Technical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 19. 5 Limitations of the Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370 19. 6 Application of CAE to the Analysis of Carbohydrate-Protein Interactions . . . . . . 371 19. 7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 20. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 20. 2 Technical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 20. 3 Sample Detection and Sample Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Autoradiography and staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Sample detection by blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Semipreparative ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 20. 4 Analysis of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Measuring sample mobilities - calculating a retardation coefficient . . . . . . . . . . . . 391 Graphical analysis of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Interpreting ACE patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Reverse ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 20. 5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 XII List of Contributors Nebojsa Avdalovic John T. Gallagher Dionex Corporation Cancer Research Campaign Department of Medical Oncology 445 Lakeside Drive University of Manchester Sunnyvale, CA 94086 Christie CRC Research Centre Klaus Biemann Wilmslow Road Department of Chemistry Manchester M20 4BX Massachusetts Institute of Technology UK Cambridge, MA 02139-4307 USA Geoffrey R.

Contents:

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.