An integrated metabolomics workflow for the quantification of sulfur pathway intermediates employing thiol protection with N-ethyl maleimide and hydrophilic interaction liquid chromatography tandem mass spectrometry

Abstract

The sulfur metabolic pathway is involved in basic modes of cellular metabolism, including methylation, cell division, respiratory oscillations and stress responses. Hence, the implicated high reactivity of the sulfur pathway intermediates entails challenges for their quantitative analysis. In particular the unwanted oxidation of the thiol group-containing metabolites glutathione, cysteine, homocysteine, γ-glutamyl cysteine and cysteinyl glycine must be prevented in order to obtain accurate snapshots of this important part of cellular metabolism. Suitable analytical methodologies are therefore needed to support studies of drug metabolism and metabolic engineering. In this work, a novel sample preparation strategy targeting thiolic metabolites was established by implementing thiol group protection with N-ethyl maleimide using a cold methanol metabolite extraction procedure. It was shown that N-ethyl maleimide derivatization is compatible with typical metabolite extraction procedures and also allowed for the stabilization of the instable thiolic metabolites in a fully (13)C-labeled yeast cell extract. The stable isotope labeled metabolite analogs could be used for internal standardization to achieve metabolite quantification with high precision. Furthermore, a dedicated hydrophilic interaction liquid chromatography tandem mass spectrometry method for the separation of sulfur metabolic pathway intermediates using a sub-2 μm particle size stationary phase was developed. Coupled with tandem mass spectrometry, the presented methodology proved to be robust, and sensitive (absolute detection limits in the low femtomole range), and allowed for the quantification of cysteine, cysteinyl glycine, cystathionine, cystine, glutamic acid, glutamyl cysteine, reduced glutathione, glutathione disulfide, homocysteine, methionine, S-adenosyl homocysteine and serine in a human ovarian carcinoma cell model.

Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer

“…The N-ethylmaleamide (NEM) derivatized, isotope labeled, [ 13 C 3 , 15 N]-cysteine-NEM, [ 13 C 3 ]-cysteine-NEM, [ 13 C 2 , 15 N]-GSH-NEM and [D 5 ]-GSH-NEM which were prepared by derivatizing the [ 13 C 3 , 15 N]-cysteine, [ 13 C 3 ]-cysteine, [ 13 C 2 , 15 N]-GSH, and [D 5 ]-GSH standards with 50 mM NEM in 10 mM ammonium formate (pH = 7.0) at room temperature (30 min) as previously described (Ortmayr et al, 2015). [ 13 C 4 , 15 N 2 ]-GSSG was prepared from the oxidation of [ 13 C 2 , 15 N]-GSH as described (Zhu et al, 2008).…”

A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome

“…Half a century ago, it was also employed for the spectrophotometric measurement of SH groups [59] . NEM and related maleimides have since been widely used for the analysis of thiols, including GSH [60] , [61] , for the prevention of GSH autoxidation [61] to stabilize human blood for later quantification of thiols/disulfides [62] ; even in a similar workflow for sulfur pathway analysis using a different chromatographic separation [63] . It has also been known for some time to react with sulfide [38] , [43] .…”

Error propagation in constraint-based modeling of Chinese hamster ovary cells

“…In fact, accurate analysis of cysteine, containing a highly reactive thiol group, requires chemical protection during extraction. A protocol using N-ethylmaleimid protection 40 of the reactive thiol improved the precision achievable by LC-MS (5-10%).…”

“…** accurate quantification of cysteine requires derivatization upon extraction. NEM derivatization can be accomplished during the extraction/protein precipitation step 40 . *** The isomers threonine/homoserine are not always separated, only by zicHILIC.…”