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. Participants will learn basic techniques for the isolation and characterization of glycoprotein and glycolipid oligosaccharides via a combination of hands-on work, lectures and demonstrations. Participants will release N- and O-linked glycans from a glycoprotein. The released glycans will be purified, derivertized and profiled by mass spectrometry. (MALDI/TOF-MS and ESI-MSn). The monosaccharide composition on a glycoprotein will be analyzed by HPEAC. Glycophingolipids will be separated by thin-layer chromatography and detection protocols will be discussed. The use of lectin blotting techniques for characterization of oligosaccharide structural features will be covered. In addition, modules on using mass spectrometry, computer modeling or NMR spectroscopy for analysis of glycoconjugates will be included. Experience with basic biochemical techniques is a prerequisite for participation. Additional Course Modules On the Friday of each of these two courses, participants can choose one of the following three modules, Mass Spectrometric (MS) Analysis of Glycoconjugates, Computational Simulations of Carbohydrate Conformation and NMR of Carbohydrates. The course participants should indicate on the course application form which module they would be interested in taking. Mass Spectrometric (MS) Analysis of Glycoconjugates: The use of mass spectrometry for the characterization of glycoconjugates will be discussed. Topics in this area will include identifying occupied glycosylation sites by LC-MS and sequencing glycoprotein glycans with sequential exoglycosidase digestions followed by MS. Laboratory demonstrations will also be performed on these two topics. Computational Simulations of Carbohydrate Conformation: The course will present a discussion of the conformational preferences of oligosaccharides. Focus will then shift to the prediction and validation of oligosaccharide structures produced computationally with molecular dynamics simulations with the AMBER/GLYCAM force field. NMR of Carbohydrates: This module will be an introduction to the application of NMR spectroscopy of polysaccharides and glycoconjugates covering: a) essential features of NMR spectra; b) protocols used to solve primary structures; c) applications and examples from the current literature, and; d) resources for information. Our four NMR instruments (300-, 500-, 600-, and 800 mHz) will be used for demonstrations during this module.