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), γ-glutamyl-cysteine (Glu-Cys), and s-adenosyl-homocysteine (AdoHcy).Tris(2-carboxyethyl) phosphine (TCEP) was used as the reducing reagent and p-hydroxymercurybenzoate (PHMB) as the derivatization reagent. Thiols were extracted from three mg of yeast using water in an ultrasonic bath. The absolute detection limits for the compounds studied were in the sub-picomole 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.

Determination of Reduced Cysteine in Oenological Cell Wall Fractions of Saccharomyces cerevisiae

“…Such dipeptides represent about 4% of GSH in yeast (20); therefore, their contribution was not taken into account also for the MP samples where the nonproteinaceous Cys thiols were never detected. The amounts of dithiol-substituted hydroquinones were not included in the calculation since their values were close to the limit of quantification (signalto-noise ratio >10).…”

Synthesis and evaluation of heparin immobilized “side-on” to polystyrene microspheres coated with end-group activated polyethylene oxide

“…1). While such total thiolation is entirely possible given the excess of carbodiimide used in this work, overestimation of thiol content by the Ellman assay is another possibility, owing to detection of Ellman-based species generated by side reaction [25] or through reaction of the -OH groups along the heparin chain in addition to the carboxylates [26].…”

A new strategy for ionization enhancement by derivatization for mass spectrometry

“…The derivatization reagents for thiols are summarized in Table 2. Reduced glutathione (GSH) could be determined by MS measurements after derivatization with 5,5 -dithio-(bis-2-nitrobenzoic) acid (DTNB) [37], iodoacetic acid [38], and p-(hydroxymercuri)benzoate [39]. Maleimide derivatization reagents react rapidly and are useful to protect the thiol group [40].…”