title: Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry.
abstract: For the first time a liquid chromatography method with high resolution mass spectrometric detection has been developed for the simultaneous determination all key metabolites of the sulfur pathway in yeast, including all thiolic (cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), γ-glutamyl-cysteine (Glu-Cys)) and non-thiolic compounds (methionine (Met), s-adenosyl-methionine (AdoMet), s-adenosyl-homocysteine (AdoHcy), and cystathionine (Cysta)). The developed assay also permits the speciation and selective determination of reduced, oxidized and protein bound fractions of all of the five thiols. Iodoacetic acid (IAA) was chosen as the derivatizing reagent. Thiols were extracted from sub-mg quantities of yeast using hot 75% ethanol. The detection limits were in the range of 1-12 nmol L(-1) for standard solution (high femotomole, absolute), except AdoMet (116 nmol L(-1)), which was unstable. In freshly harvested yeast, most of the thiols were in the reduced forms and low levels of protein-bound GSH and Glu-Cys were found. In a selenium enriched yeast, the thiols were mainly in the oxidized forms, and a significant amount of protein-bound Cys, HCys, GSH, Cys-Gly and Glu-Cys were found. The method was also applied to the metabolic study of the adaptive response of Saccharomyces cerevisiae to hydrogen peroxide, cadmium, and arsenite, and the change in concentration of thiols in the sulfur pathway was monitored over a period of 4h.
authors.affiliation: Institute for National Measurement Standard, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Yulan
authors.initials: Y
authors.lastname: Rao
authors.forename: Margaret
authors.initials: M
authors.lastname: McCooeye
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Mester
mesh_terms: D018053:Arsenites / Q000633:toxicity; D002104:Cadmium / Q000633:toxicity; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity; D019807:Iodoacetic Acid / Q000737:chemistry; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D012643:Selenium / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000378:metabolism
chemical_list: D018053:Arsenites; D013438:Sulfhydryl Compounds; D002104:Cadmium; D006861:Hydrogen Peroxide; D005978:Glutathione; D012643:Selenium; D003545:Cysteine; C015001:arsenite; D019807:Iodoacetic Acid