This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Project 2 is focusing on quantitating components within single or very small numbers (~ 10) of cells that have been labeled with levels of 14C. Our approach will develop protocols to label cells with 14C so that molecular components of a chosen cell possess a specific isotopic signature. Individual or very small numbers of labeled cells will then be placed in a pool of similar cells that contain natural isotope concentrations. Once lysed, cellular components will be speciated chromatographically and isolated metabolites identified by mass spectrometry. AMS will provide quantification of the 14C isotope signal in the isolated metabolite. The quantified 14C isotope signal arises predominantly from the metabolites of the labeled cell in the isolated component, while the metabolite components from the unlabeled cells provide reliability of speciation and identification of the eluted fractions. In principle this approach could provide quantitative metabolite profiles of single or small numbers of cells while using accepted or advancing laboratory protocols for isolation and identification of metabolites. The relatively large amount of sample material mitigates concerns about quantitative sample recovery during speciation. As a common theme for this work we are studying the budding yeast Saccharomyces cerevisiae as a model eukaryotic cell. Levels of nicotinamide adenine dinucleotides appear to play important roles during calorie restriction (CR) in extending replicative lifespan in S. cerevisiae. A better understanding of these metabolite levels in S. cerevisiae during CR and aging may lead to a better understanding of molecular mechanisms underlying age-related diseases.
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