Sensitive Methods for Glycoconjugate Analysis

Trustees Of Indiana University, Bloomington IN

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Abstract

DESCRIPTION (provided by applicant): Support continuation is being requested for a highly productive research in analytical glycobiology at Indiana University (IU). High-sensitivity quantitative measurements of glycoproteins and positive identification of their constituent glycan components are very important to many areas of biomedical research, particularly in a search for disease biomarkers. While our research during the last several years has made it possible to profile quantitatively N-glycans at considerably greater sensitivities than ever before, the proposed methodological improvements will focus increasingly on covering O-glycans using (a) greatly improved techniques of glycan release/MALDI mass spectrometry (MS); (b) derivatization and mass-shift strategies; and (c) profiling smaller O-glycans through GC/MS of permethylated derivatives. Additionally, significant improvements in preconcentration of trace glycoproteins from physiological fluids and tissues will be sought through the use of (a) surface-immobilized lectins; (b) surface-immobilized Protein L and selected antibodies; and (c) molecular size-based fractionation through novel capillary technologies. The surface- immobilized glycoprotein preconcentrators will utilize new types of highly porous silica materials prepared recently in a collaborating IU laboratory. New lectin types will augment this approach, focusing primarily on highly sialylated and hyperfucosylated glycans (both N- and O-types) that our laboratory has targeted as parts of cancer biomarkers under a complementary NCI grant. Our preliminary results with surface- immobilized Protein L provide indications that such procedures could rapidly sort out different immunoglobulin glycosylation patterns in very small volumes (few microliters) of physiological fluids. Alternatively, selected surface-immobilized antibodies to acute phase glycoproteins can be used to preconcentrate the fractions of diagnostic/prognostic interest. Properly treated extracts of tissues and cancer cell lines will als be investigated through a set of optimized procedures that will augment the analytical microscale methodologies developed for blood serum and plasma. With our laboratory's mass-spectrometric instrumentation and expertise in place, we will continue our emphasis on optimal biological sample treatment, glycoprotein fractionation, and preconcentration at microscale.