SEPARATION AND CHARACTERIZATION OF GLYCOPROTEIN AND GLYCOLIPID 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. In this course participants learned basic techniques for the isolation and characterization of oligosaccharides via a combination of hands-on work, lectures and demonstrations. Mixtures of oligosaccharides or glycopeptides derived from glycoproteins were resolved into individual components by chromatographic procedures such as size-exclusion, HPLC, lectin affinity chromatography, and/or polyacrylamide gel electrophoresis. Glycosphingolipids were separated by thin-layer chromatography, and detection protocols were discussed. The use of lectin blotting techniques for characterization of oligosaccharide structural features were covered. Other topics which were 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 were included (see descriptions below). Experience with basic biochemical techniques was a prerequisite for participation. Additional Course Modules Mass Spectrometric (MS) Analysis of Glycoconjugates: The use of mass spectrometry for the characterization of glycoconjugates was discussed. Topics included in this area included identifying occupied glycosylation sites by LC-MS and sequencing glycoprotein glycans with sequential exoglycosidase digestions followed by MS. Laboratory demonstrations were performed on these two topics. NMR of Carbohydrates: This module was an introduction to the application of NMR spectroscopy of 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) were used for demonstrations during this module.