Optimized Protocol for the In Situ Derivatization of Glutathione with N-Ethylmaleimide in Cultured Cells and the Simultaneous Determination of Glutathione/Glutathione Disulfide Ratio by HPLC-UV-QTOF-MS

Abstract

Glutathione (GSH) and glutathione disulfide (GSSG) are commonly used to assess the oxidative status of a biological system. Various protocols are available for the analysis of GSH and GSSG in biomedical specimens. In this study, we present an optimized protocol for the in situ derivatization of GSH with N-ethylmaleimide (NEM) to prevent GSH autooxidation, and thus to preserve the GSH/GSSG ratio during sample preparation. The protocol comprises the incubation of cells in NEM containing phosphate buffered saline (PBS), followed by metabolite extraction with 80% methanol. Further, to preserve the use of QTOF-MS, which may lack the linear dynamic range required for the simultaneous quantification of GSH and GSSG in non-targeted metabolomics, we combined liquid chromatographic separation with the online monitoring of UV absorbance of GS-NEM at 210 nm and the detection of GSSG and its corresponding stable isotope-labeled internal standard by QTOF-MS operated with a 10 Da Q1 window. The limit of detection (LOD) for GS-NEM was 7.81 µM and the linear range extended from 15.63 µM to 1000 µM with a squared correlation coefficient R2 of 0.9997. The LOD for GSSG was 0.001 µM, and the lower limit of quantification (LLOQ) was 0.01 µM, with the linear (R2 = 0.9994) range extending up to 10 µM. The method showed high repeatability with intra-run and inter-run coefficients of variation of 3.48% and 2.51% for GS-NEM, and 3.11% and 3.66% for GSSG, respectively. Mean recoveries of three different spike-in levels (low, medium, high) of GSSG and GS-NEM were above 92%. Finally, the method was applied to the determination of changes in the GSH/GSSG ratio either in response to oxidative stress in cells lacking one or both monocarboxylate transporters MCT1 and MCT4, or in adaptation to the NADPH (nicotinamide adenine dinucleotide phosphate) consuming production of D-2-hydroxyglutarate in cells carrying mutations in the isocitrate dehydrogenase genes IDH1 and IDH2.

In situ synthesis of chiral AuNCs with aggregation-induced emission using glutathione and ceria precursor nanosheets for glutathione biosensing

“…Using this new MRT strategy, an eco-friendly, selective, biocompatible and label-free AIE probe was established for the sensitive sensing of GSH with a limit of detection (LOD) of 1.02 µM.Moreover, this switch-on luminescent nanoplatform of the conjugate probe of Au-thiolate/Ce-based NSs was successfully applied for the selective and reliable GSH detection in human serum samples. liquid chromatography (HPLC) 11,12 and surface-enhanced Raman scattering (SERS), 13,14 require high-cost devices, lengthy sensing operation as well as well-trained technicians.Recently, AuNCs have witnessed great progress as nanoprobes for sensing of various biological molecules. 15 Specifically, thiolate-stabilized AuNCs are one of the most frequently applied AuNCs for photoluminescence sensing due to their inherent appealing features, including facile and eco-friendly synthesis, ultrasmall size (<3.0 nm), size-dependent photoluminescence (PL), anti-photobleaching and excellent biocompatibility.…”

“…Moreover, this switch-on luminescent nanoplatform of the conjugate probe of Au-thiolate/Ce-based NSs was successfully applied for the selective and reliable GSH detection in human serum samples. liquid chromatography (HPLC) 11,12 and surface-enhanced Raman scattering (SERS), 13,14 require high-cost devices, lengthy sensing operation as well as well-trained technicians.…”

Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS

“…All regression coefficients (R 2 ) were greater than 0.99. The limit of quantification differed for each analyte and ranged from 0.01 ng/mL for GSH to 5 ng/mL for GSSG, indicating that the sensitivity of this method is comparable or superior to that of other methods for the analytes investigated here [ 20 , 21 , 30 , 31 , 32 , 33 ].…”

“…It has been reported that the derivatization of thiols with maleimide, including both NEM and NPM (N-(1-pyrenyl) maleimide), will generate diastereomers, some of which can be separated by HPLC, resulting in two peaks [ 6 , 21 , 23 , 28 , 29 ]. Such diastereomer peaks were also observed in this study.…”

“…The ability to quickly and conveniently profile thiol redox pathways in response to oxidative insult could further elucidate pathological mechanisms and, consequently, uncover potential therapeutic targets [ 16 , 17 , 18 , 19 ]. However, previous methods fall short in one or more important areas: compared to HPLC-UV or -FLD, LC-MS methods are capable of simultaneously measuring thiols (or their derivatives) and their corresponding disulfides with less sample workup, but most focus exclusively on the determination of GSH and GSSG [ 20 , 21 , 22 , 23 , 24 ]. One notable exception is a robust LC-MS/MS method developed by Sutton et al for analyzing the thiol redox metabolome in blood, urine, and saliva [ 6 ].…”