This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for 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 fuller 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. To measure metabolites from individual 14C labeled cells with AMS, it is important that robust methods be available to label and to manipulate such cells. We initially cultured yeast in 14C labeled glucose to label yeast cells but as nicotinic acid is a precursor of intracellular pyridine dinucleotides and is present in the culture medium levels of [14C]-NAD and [14C]-NADH in the cultured yeast were low. Yeast are now grown in synthetic complete medium containing [14C]-nicotinic acid enabling synthesis of [14C]-NAD and 14C]-NADH. Use of [14C]-nicotinic acid ensures greater labeling of NAD and NADH than labeling with glucose and 14C contents of labeled yeast are typically around 70 amol. We have performed 14C measurements on 4 strains of yeast to demonstrate the feasibility of this labeling scheme. Following culture, [14C]-labeled yeast cells are removed from the labeled medium, washed and placed in a pool of ~ 108 similar yeast cells that contain natural carbon isotope concentrations. Once lysed, cellular metabolites including NAD, NADH are extracted from this pool of cells using a simultaneous redox and energy state analysis extraction protocol and speciated by High Performance Liquid Chromatography (HPLC). We have refined our metabolite extraction and HPLC protocols and now have a robust HPLC protocol for determining NAD and NADH in pools of yeast. Accelerator Mass Spectrometry is used to quantitate the 14C isotope signal in the isolated HPLC fractions and we are currently utilizing the technique to measure NAD and NADH in populations of ~1000 cells. In the coming year we anticipate being able to reduce the number of cells to the range of 1 -10.
Showing the most recent 10 out of 125 publications
