| Abstract
| - Metabolomics, i.e., the global analysis of cellular metabolites, is becoming a powerful tool for gaining insights intobiological functions in the postgenomic context. However,absolute quantitation of endogenous metabolites in biological media remains an issue, and available technologiesfor the analysis of metabolome still lack robustness andaccuracy. We describe here a new method based on liquidchromatography−mass spectrometry and 15N uniformmetabolic labeling of Saccharomyces cerevisiae foraccurate and absolute quantitation of nitrogen-containingcell metabolites in metabolic profiling experiments. As aproof of concept study, eight sulfur metabolites involvedin the glutathione biosynthesis pathway (i.e., cysteine,homocysteine, methionine, γ-glutamylcysteine, cystathionine, reduced and oxidized forms of glutathione, andS-adenosylhomocysteine) were simultaneously quantified.The analytical method has been validated by studies ofstability, selectivity, precision, and linearity and by thedetermination of the limits of detection and quantification.It was then applied to the analysis of extracts fromcadmium-treated yeasts. In these conditions, the intracellular concentrations of most of the metabolites involvedin the glutathione biosynthesis pathway were increasedwhen compared to control extracts. These data correlatewith previous proteomic results and also underline theimportance of glutathione in cadmium detoxication.
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