TECHNIQUES FOR CHARACTERIZATION OF CARBOHYDRATE STRUCTURE OF POLYSACCHARIDES

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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. Techniques for Characterization of Carbohydrate Structure of Polysaccharides This is a one-week course which covers the techniques of glycosyl-residue and glycosyl-linkage composition analyses using gas-liquid chromatography/mass spectrometry (GC/MS). The hands-on work includes the derivatization of sugar samples to make alditol acetates and trimethylsilyl derivatives and the production of partially methylated alditol acetates that the trainees analyze by GC/MS. Lectures and demonstrations cover such techniques for structural analysis MALDI-MS ans ESI-MS mass spectrometry, ^1 H-NMR spectroscopy, and methods for the separation and purification of monosaccharides and oligosaccharides using Metrohm peak HPLC and other chromatographic techniques. Additional lectures are also given on specific topics such as Chemical synthesis of oligosaccharides and isolation and purification of polysaccharides This course covers the techniques of glycosyl residue (composition) and glycosyl linkage (methylation) analyses using gas-liquid chromatography/mass spectrometry. Participants perform analyses of purified oligosaccharides. Lectures and demonstrations cover other techniques for structural analysis. In addition, a choice of modules either on using mass spectrometry or NMR spectroscopy for analysis of glycoconjugates is included (see descriptions below). Experience with basic biochemical techniques is a prerequisite for participation. 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. Computational Simulations of Carbohydrate Conformation: The course presents a discussion of the conformational preferences of oligosaccharides. Focus shifts to the prediction and validation of oligosaccharide structures produced computationally with molecular dynamics simulations with the AMBER/GLYCAM force field.