This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The research area of Cell Biology focuses on gaining an understanding of both the composition and function of individual cells, how cells change over their lifetime and in response to signals from their environment as well as determining how cells form a whole organism. Biochemical studies of cells and their contents depend on collecting, defining, identifying, and quantifying the biomolecules responsible for a certain function, structure, or biochemical signal. Such well-defined biomolecular samples often contain little material. To address this paucity of material, a variety of sensitive instrumentation, including flow cytometers and capillary electrophoresis (CE) systems, are available to provide sensitivity for studying these materials. Such techniques typically employ chemical reactions to enable fluorescent labeling and conventional or laser-induced fluorescence for detection. While such methods have detection sensitivities as low as several zeptomole for proteins, not all moieties within a cell can be fluorescently labeled and quantitation often relies on molecular properties of the analyte. Isotope labeling strategies provide an alternative approach to protocols that employ fluorescence detection. The isotope label can be analytically measured independent of molecular structure to quantitate the amount of a substance. 13C and 14C are frequently used in isotope labeling experiments primarily because they are isotopes of an element naturally present in almost all molecules of biological interest. Cells can be cultured in enriched 13C or 14C media so that, in principle, all cellular constituents are uniformly labeled. This means that all compounds within the cell can be efficiently labeled. AMS, owing to its ability to measure small quantities of material at high sensitivity and with accurate analytical quantitation, is well suited to make valuable contributions to cell biology. Successful use of AMS for cell biology will not only require adequate sample preparation methods, but also will require that the biological aspects of the experiments be designed and conducted for compatibility with AMS analysis. This project will develop methods: 1) to routinely couple cell biology experiments to AMS and;2) to quantitate components within cells that have been labeled with 14C. Emphasis will be placed on the development of separatory methods to speciate metabolites and molecules from pools of cells for accurate quantification by AMS. Specifically, we will develop robust methods to: 1) Isolate sub-cellular fractions and speciate individual metabolites from pools of cells. 2) Couple flux balance analysis to AMS. 3) Couple AMS to quantitative measurement of post translational modifications. 4) Couple AMS to cell turnover in human tissues using the 14C bomb curve.
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