SEPARATION AND CHARACTERIZATION OF GLYCOPROTEIN AND GLYCOLIPID OLIGOSACCHARIDES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Separation and Characterization of Glycoprotein and Glycolipid Oligosaccharides. 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". 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.