Accurate redox state indication by in situ derivatization with N-ethylmaleimide - Profiling of transsulfuration and glutathione pathway metabolites by UPLC-MS/MS.

Background: Reduced and oxidized glutathione play an important role for the intracellular detoxification of reactive oxygen species. The iron-dependent formation of such reactive oxygen species in conjunction with the inhibition of the redox-balancing enzyme glutathione peroxidase 4 underlie an imbalance in the cellular redox state, thereby resulting in a non-apoptotic form of cell death, defined as ferroptosis, which is relevant in several pathologies.

Methods: Here we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based method providing the accurate quantification of 12 glutathione pathway metabolites after in situ derivatization with N-Ethylmaleimide (NEM). The method was validated regards linearity, recovery and accuracy as well as precision. The assay includes glutathione and its oxidized form glutathione disulfide. Furthermore, the related precursors cysteine, cystine, glutamic acid, γ-glutamylcysteine and cysteinylglycine, biomarkers of protein crosslinking such as cystathionine and lanthionine, as well as metabolites of the transsulfuration pathway, methionine, homocysteine and serine are simultaneously determined.

Results: Twelve glutathione pathway metabolites were simultaneously analyzed in four different human cell line extracts within a total LC run time of 5.5 min. Interday coefficients of variation (1.7 % to 12.0 %), the mean observed accuracy (100.0 % ± 5.2 %), linear quantification ranges over three orders of magnitude for all analytes and sufficient metabolite stability after NEM-derivatization demonstrate method reliability. Immediate derivatization with NEM at cell harvesting prevents autooxidation of glutathione, ensures accurate results for the GSH/GSSG redox ratio and thereby allows interpretation of cellular redox state.

Conclusion: The described UPLC-MS/MS method provides a sensitive and selective tool for a fast and simultaneous analysis of glutathione pathway metabolites, its direct precursors and related compounds. Assay performance characteristics demonstrate the suitability of the method for applications in different cell cultures. Therefore, by providing glutathione related functional metabolic readouts, the method enables investigations in mechanisms of ferroptosis and alterations in oxidative stress levels in several pathophysiologies.