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ARS Home » Plains Area » Grand Forks, North Dakota » Grand Forks Human Nutrition Research Center » Dietary Prevention of Obesity-related Disease Research » Research » Publications at this Location » Publication #351362
Title: Quantitation of glutathione, glutathione disulfide and protein-glutathione mixed disulfides by high performance liquid chromatography-tandem mass spectrometry

Author
item Bukowski, Michael
item Picklo, Matthew

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 3/13/2018
Publication Date: 5/9/2019
Citation: Bukowski, M.R., Picklo, M.J. 2019. Quantitation of glutathione, glutathione disulfide and protein-glutathione mixed disulfides by high performance liquid chromatography-tandem mass spectrometry. Methods in Molecular Biology. https://doi.org/10.1007/978-1-4939-9187-7_12.
DOI: https://doi.org/10.1007/978-1-4939-9187-7_12

Interpretive Summary: Glutathione is a potent antioxidant in plant and animal cells that can form complexes with proteins through a process called glutathionylation, which protects the protein from oxidative damage. Increasingly glutathionylation has been found to act as switch to regulate the function of proteins, turning them on or off, including proteins related to the development of obesity and inflammation. The measurement of glutathione content in protein is important in determining both the level of oxidative damage in a biological system, as well as which biological pathways are activated by this switch. Previously published methods routinely overestimated protein glutathionylation because they cannot differentiate free glutathione species from protein-bound glutathione. Our work uses liquid chromatography coupled mass spectrometry to differentiate protein-bound glutathione from these other glutathione species. This chapter is a step-by-step protocol for our method will benefit life scientists that study protein function in agricultural, nutrition, and biomedical research.

Technical Abstract: Protein-gluthione mixed disulfides (PSSG) are an important redox-sensitive post-translational modification. Quantitation of protein-gluthione mixed disulfides (PSSG) is achieved by the reduction of the disulfide bond to liberate glutathione (GSH); however, this method leaves the assay susceptible to contamination by cytosolic GSH and glutathione disulfide (GSSG) captured during protein precipitation. The method herein describes a work flow in which protein from mouse liver is precipitated and adventitious GSH contamination is removed by reaction with N-ethylmaleimide. The sample is divided into two equal aliquots, a control aliquot that allows for direct quantitation of adventitious GSSG and a chemically reduced aliquot that contains GSH from both the GSSG and PSSG disulfides. Determining the concentration of adventitious GSSG allows for correction of the latter value to provide an accurate assay of PSSG. This assay also provides quantitation of cytosolic GSH and GSSG.