This proposal describes a highly interdisciplinary effort by a team of chemists, biologists, engineers, and information scientists who will collaboratively develop a new set of tools for characterizing and recognizing temporal changes in the metabolome of model organisms. Three sets of tool will be developed; those that enhance the quantification of changes in the metabolome, instrumentation that augments throughput by either massively parallel analysis or differential analysis, and separation materials and techniques that improve sampling and resolution of metabolites. Using a collection of gene knockout mutants in Escherichia coli and a knockin mutant in Saccharomyces cerevisiae these global analysis tools will be used to identify metabolic nodes, control networks, and linkages between the metabolome and genome. Quantification efforts will focus on the preparation and use of stable isotope coded reagents for determining relative concentration, absolute amounts, and metabolic flux of metabolites. Mass spectral and nuclear magnetic resonance instruments and tools will play a critical role in this proposal. A revolutionary new mass spectrometer will be built that is a miniature array of 96 mass analyzers with each analyzer monitoring a small mass range continuously. As part of our effort to monitor metabolic flux in vivo we are designing and building miniaturize flow cells for NMR studies that will allow 2-4 samples to be monitored simultaneously. Miniaturized flow cells along with sample concentrators will be of critical importance in the global analyses of metabolites. In addition we are developing tools that will provide the first study of genes that impact utilization of mineral ions by cells. Finally universal separation media are being developed for liquid phase separations of metabolites.
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