Biomolecular quantitation in single cells is needed to investigate cellular asymmetric processes and intercellular variability, and to validate the mathematical models of metabolic and regulatory processes against experiments. Conventional biochemistry typically measures the average concentration of analytes from large numbers of cells (~109). This does not address the problems associated with many fundamental biological processes which occur within an individual cell such as aging, cell differentiation, carcinogenesis, cell-cell communication, etc. Therefore, we are developing methods for quantitative and concurrent measurement of intracellular metabolites from single cells based on 14C-labeling experiments and accelerator mass spectrometry (AMS). Cells are grown on 14C-enriched growth media for multiple generations so that, in principle, all cellular constituents are eventually labeled whether they are structural constituents or metabolic intermediates and products. Then, single cells grown on 14C-labeled media are rinsed and mixed with a pool of unlabeled cells (~10 7) grown under the same environmental conditions to minimize the concerns associated with speciation and sample recovery. The intracellular metabolites are extracted and separated based on established protocols via nano-HPLC, and fractions that represent different metabolites are processed for AMS analysis. The intracellular metabolite profiles from single yeast (Saccharomyces cerevisiae) cells based on our developed method have been measured. The age of these specific cells are determined by microscopically observing the number of times the chosen cells produce a daughter cell (replicative age). The method will be applied to understanding cellular metabolism as a function of age. This work was performed in part under the auspices of the U.S. Department of Energy by University of California Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48.
See more of AMS in Low Dose Biosciences Posters
See more of The 10th International Conference on Accelerator Mass Spectrometry (September 5-10, 2005)