This proposal describes continuation of our research in developing highly sensitive and informative analytical methodologies beneficial for glycobiology research. Detailed structural information is now routinely required from minute quantities of complex glycoconjugates through an effective combination of microisolation, microcolumn separation and mass spectrometry. This includes oligosaccharide sequences, branching and linkages, all to be achieved at high sensitivity. The major methodological hurdles for submicrogram analysis of both N- and O-glycans were solved under the present grant. Efforts are now focused on the development of on-line microscale isolation of glycoprotein and enzymatic sequencing, labeling, separation and tandem MS analyses of N- and O-glycans derived from biological samples to facilitate better understanding of the biology underlying human diseases. Analytical advancements are being thought through the development of numerous methodologies including: (1) lectin microcolumns for on-line trapping of glycoproteins/glycopeptides, (2) endo-and exoglycosidase microreactors for faster and more efficient enzymatic characterization of N-glycans, (3) on-line per-O-methylation and coupling to LC/MSMS, (4) MS-based technology for the analysis of sialylated glycans, (5) derivatization of glycans to enhance separation and ionization, and (6) hydrophilic porous polymer monolith LC columns. The potential of the different proposed methodologies will be demonstrated in the areas of cancer research and alcohol abuse. The proposed methodologies will be employed to construct comprehensive glycomic/glycoproteomic maps for (a) biological samples (i.e., blood, tissue, breast duct lavage) obtained from cancer patients, and (b) brain and liver tissues obtained from rats with chronic alcohol exposure. The proposed analytical methodologies, which should result in pushing the sensitivity of current methodologies by several orders of magnitude, will prompt improved prevention, diagnosis, and treatment in the area of cancer research. They will also advance better understanding of alcohol abuse at the molecular level.
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