Future advances in biochemical research and biomedical sciences are greatly dependent upon analytical separation methods. Among the recent important developments in separation science, microcolumn liquid chromatography and capillary electrophoresis are uniquely suited to deal wit exceedingly complex biological mixtures ad the challenges of extremely sensitive detection and structural characterization. The present proposal is targeted toward significant improvements in the general capabilities of these separation methods for structural studies and analysis of the most important biological macromolecules, proteins and ribonucleic acids. While microcolumns will be employed in the separation and microisolation of biomolecules, their coupling with laser- based fluorescence detectors ensures extremely high detection sensitivity for degradation products of the biomacromolecules, such as amino acids, peptides, ad nucleotides., The proposed developments will enable analysis in the range of 0-15 - 10-18 gram quantities. Consequently, the important techniques of sequencing and peptide mapping will make it possible to characterize roughly 10,000-times smaller amounts of biomolecules than is currently feasible. These advances will enable work with extremely small volumes of biological fluids, approaching detection at the level of single cells. Whenever feasible, components of separated mixtures will be identified by mass spectrometry, which couples conveniently with the miniaturized columns. Extremely high sensitivities obtained through laser-based detection are primarily due to unique synthetic designs of fluorogenic reagents in this laboratory. These powerful new bioanalytical techniques will be applied to tow problems of biomedical significance: (a) studies of interactions between model proteins and aldehydic neurotoxins and (b) characterization of the polypeptide carriers of reproduction-related pheromones in rodents.
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