化学
谷胱甘肽
代谢组
代谢组学
代谢途径
质谱法
半胱氨酸
色谱法
代谢物
胱硫醚β合酶
生物化学
新陈代谢
酶
作者
Alexandra Lafaye,Jean Labarre,Jean‐Claude Tabet,Eric Ézan,Christophe Junot
摘要
Metabolomics, i.e., the global analysis of cellular metabolites, is becoming a powerful tool for gaining insights into biological functions in the postgenomic context. However, absolute quantitation of endogenous metabolites in biological media remains an issue, and available technologies for the analysis of metabolome still lack robustness and accuracy. We describe here a new method based on liquid chromatography-mass spectrometry and (15)N uniform metabolic labeling of Saccharomyces cerevisiae for accurate and absolute quantitation of nitrogen-containing cell metabolites in metabolic profiling experiments. As a proof of concept study, eight sulfur metabolites involved in the glutathione biosynthesis pathway (i.e., cysteine, homocysteine, methionine, gamma-glutamylcysteine, cystathionine, reduced and oxidized forms of glutathione, and S-adenosylhomocysteine) were simultaneously quantified. The analytical method has been validated by studies of stability, selectivity, precision, and linearity and by the determination of the limits of detection and quantification. It was then applied to the analysis of extracts from cadmium-treated yeasts. In these conditions, the intracellular concentrations of most of the metabolites involved in the glutathione biosynthesis pathway were increased when compared to control extracts. These data correlate with previous proteomic results and also underline the importance of glutathione in cadmium detoxication.
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