Determination of Thiols in Yeast by HPLC Coupled with LTQ-Orbitrap Mass Spectrometry after Derivatization with p-(Hydroxymercuri)benzoate

Abstract

A liquid chromatography method with mass spectrometric detection has been developed for the simultaneous determination of six thiols in the sulfur metabolic pathway, including cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), gamma-glutamyl-cysteine (Glu-Cys), and S-adenosyl-homocysteine (AdoHcy). Tris(2-carboxyethyl)phosphine (TCEP) was used as the reducing reagent and p-(hydroxymercuri)benzoate (PHMB) as the derivatization reagent. Thiols were extracted from 3 mg of yeast using water in an ultrasonic bath. The absolute detection limits for the compounds studied were in the subpicomole range. It was found that AdoHcy, Cys, GSH, Cys-Gly, Glu-Cys, and very little HCys were present in the selenium-enriched yeast sample studied, and GSH, Glu-Cys, very little AdoHcy, Cys, and Cys-Gly were present in three bakery yeasts.