The purpose is to provide investigators with structural analyses of oligosaccharides isolated from mouse cells and tissues using a combination of methods. Isolation/fractionation of glycoconjugates will be done initially at the Core with the involvement of appropriate project personnel. In the future, if the number of samples to be analyzed increases beyond the capabilities of one technician, the project personnel will be trained to perform this step, and Core personnel will perform subsequent analyses. Some facilities and technologies (e.g., those for release and profiling of oligosaccharides, analysis of sialic acids, etc) are already available in the existing UCSD Glycobiology Core service under Dr. Manzi's supervision. Sialic acid type and amount will be initially determined (see below). Glycopeptides will be prepared (see below) and the approximate ratio of different termini determined on the mixture. Oligosaccharides will then be obtained from the glycopeptides, and after initial profiling, their characteristics evaluated by enzymatic treatments combined with HPLC analysis (see below). Apart from proving that the predicted change in glycosylation has been obtained, it is important to know if additional changes occur. Because of this, characterization by physico-chemical methods will be done on those oligosaccharides that chant in content. The present level of sensitivity of the physico- chemical methods required for the structural analysis of complex glycans(i.e. NMR spectroscopy, mass spectroscopy, etc.,) should allow complete or near-to-complete structural characterization of complex oligosaccharides in the nanomole range. The projects will be capable of generating sufficient amounts of tissues/cells that would allow the Core to isolate/purify nanomole amounts of pure glycans.(see details below) It is obvious that not all of the interesting oligosaccharides generated by the projects in this proposal will be fully characterized within the time and funding allocated to this Program. We will focus on those that are recognized as critical for the development of the corresponding project. Some aspect of the work involved in completely characterizing a complex oligosaccharide structure what require additional collaborative agreements between the individual investigators and the Core director. We predict that many exciting new avenues will be opened by the research proposed here.
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