This proposal describes research for the development, evaluation, and application of new technology which will allow aqueous biochemical and biological systems to be analyzed directly and on-line by mass spectrometry. Much of the work will center around the continuous-flow FAB probe, a new method for the introduction of aqueous solutions into a mass spectrometer. Work is proposed for implementing improvements to the operational characteristics of this device, including increasing its stability, further decreasing memory effects, improving quantitative accuracy, and improving sensitivity. A major portion of the proposal describes the development of methods and applications for the use of the CF-probe for the on-line monitoring, by continuous-flow or by flow-injection techniques, of biochemical and biological systems. Initial work will focus on enzymic reactions using specific proteases, including the determination of enzyme specificity and catalytic properties with natural substrates, measurement of kinetic parameters, and substrate processing. Research is also proposed for the development and application of HPLC and electrophoresis techniques as integrated processes for the mass spectrometer. The HPLC/MS development will concentrate on microbore and capillary techniques because the low flow rates and relatively high analyte concentrations, and will mainly focus on the analysis of enzymic digests of proteins and glycosidase catalyzed hydrolyses of oligosaccharides. These studies will also involve on-line use of immobilized exopeptidases for sequence analysis of separated FAB for the production of ions, providing a method for the separation of charged species in a solution with mass specific analysis. The research program described herein will provide innovative approaches through which mass spectrometry will play a major role in studies designed to gain insight into the intricacies of biological processes at the molecular level.
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