SEPARATION AND CHARACTERIZATION OF GLYCOCONJUGATE OLIGOSACCHARIDES

University Of Georgia, Athens GA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Separation and Characterization of Glycoconjugate Oligosaccharides (one-week course) This is a one-week course that covers basic techniques for the isolation and characterization of oligosaccharides, including colorimetric quantitation of sugars, chromatographic separation of oligosaccharide mixtures by HPLC, Metrohm peak high-pH anion-exchange chromatography for monosaccharide composition analysis, enzymatic release of oligosaccharides from a glycoconjugate, separation of glycolipids by thin-layer chromatography and detection by overlay assay, and endo- and exoglycosidase treatment of oligosaccharides. The use of lectin blotting techniques for characterization of oligosaccharide structural features are covered. Other topics included are conjugation of oligosaccharides with fluorescent tags, capillary electrophoresis and monosaccharide composition analysis. In addition, modules on using mass spectrometry and/or NMR spectroscopy for analysis of glycoconjugates are included (see descriptions below). Experience with basic biochemical techniques is a prerequisite for participation. In addition to CCRC faculty, other speakers were invited to help teach the course. Dr. Alfred Merrill from Georgia Institute of Technology gave a lecture on "Structural Analysis of Glycolipids by Mass Spectrometry". Dr. Jon Amster from the Chemistry Department at University of Georgia also gave a lecture on "Analysis of oligosaccharides by FT-MS. Additional Course Modules Mass Spectrometric (MS) Analysis of Glycoconjugates: The use of mass spectrometry for the characterization of glycoconjugates is discussed. Topics in this area include identifying occupied glycosylation sites by LC-MS and sequencing glycoprotein glycans with sequential exoglycosidase digestions followed by MS. Laboratory demonstrations are performed on these two topics. NMR of Carbohydrates: The NMR module consists of an introduction to the application of NMR spectroscopy to polysaccharides and glycoconjugates covering (i) essential features of NMR spectra, (ii) protocols used to solve primary structures, (iii) applications and examples from the current literature, and (iv) resources for information. Our four NMR instruments (300-, 500-, 600-, and 800-MHz) are used for demonstrations during this module.