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  • An UPLC-MS/MS Assay to Measure Glutathione as Marker for Oxidative Stress in Cultured Cells.

An UPLC-MS/MS Assay to Measure Glutathione as Marker for Oxidative Stress in Cultured Cells.

Metabolites (2019-03-08)
Katharina Herzog, Lodewijk IJlst, Arno G van Cruchten, Carlo W T van Roermund, Wim Kulik, Ronald J A Wanders, Hans R Waterham
ABSTRACT

Oxidative stress plays a role in the onset and progression of a number of diseases, such as Alzheimer's disease, diabetes and cancer, as well as ageing. Oxidative stress is caused by an increased production of reactive oxygen species and reduced antioxidant activity, resulting in the oxidation of glutathione. The ratio of reduced to oxidised glutathione is often used as a marker of the redox state in the cell. Whereas a variety of methods have been developed to measure glutathione in blood samples, methods to measure glutathione in cultured cells are scarce. Here we present a protocol to measure glutathione levels in cultured human and yeast cells using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC⁻MS/MS).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DL-Buthionine-(S,R)-sulfoximine
Sigma-Aldrich
Dimethyl sulfoxide solution, 50 wt. % in H2O
Sigma-Aldrich
N-Ethylmaleimide, crystalline, ≥98% (HPLC)
Sigma-Aldrich
L-Glutathione oxidized, ≥98% (HPLC)
Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
Menadione, crystalline
Milli-Q
Milli-Q® IQ 7000 Ultrapure Water Purification System, output: type 1 water (18.2 MΩ·cm), the most advanced Milli-Q® ultrapure (Type 1) water system that is intelligent, intuitive, and green.
Sigma-Aldrich
Glutathione-(glycine-13C2,15N) trifluoroacetate salt, ≥99 atom % 13C, ≥98 atom % 15N, ≥95% (CP)