Liquid chromatography-mass spectrometry and 15N metabolic labeling for quantitative metabolic profiling.
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.
Lafaye
Alexandra
A
CEA, Service de Pharmacologie et d'Immunologie, DSV/DRM, 91191 Gif-sur-Yvette Cedex, France.
Labarre
Jean
J
Tabet
Jean-Claude
JC
Ezan
Eric
E
Junot
Christophe
C
D002104:Cadmium / Q000494:pharmacology; D002853:Chromatography, Liquid / Q000379:methods*; D005978:Glutathione / Q000096:biosynthesis; D007553:Isotope Labeling / Q000379:methods*; D016014:Linear Models; D008967:Molecular Biology / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013455:Sulfur / Q000378:metabolism
D009587:Nitrogen Isotopes; D002104:Cadmium; D013455:Sulfur; D005978:Glutathione