|BUCKLIN, CHRISTOPHER - University Of North Dakota|
Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2014
Publication Date: 1/15/2015
Publication URL: http://handle.nal.usda.gov/10113/59914
Citation: Bukowski, M.R., Bucklin, C., Picklo, M.J. 2015. Quantitation of protein S-glutathionylation by liquid chromatograph-tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide. Analytical Biochemistry. 469:54-64.
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 method will benefit life scientists that study protein function in agricultural, nutrition, and biomedical research.
Technical Abstract: Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein-glutathione mixed disulfides (PSSG) are commonly quantified by the reduction of the disulfide and detection of the resultant glutathione species. This methodology is susceptible to contamination by free, unreacted cellular glutathione (GSH) species, which are present in 1000-fold greater concentration. An LCMS-based method was developed for quantification glutathione and glutathione disulfide (GSSG) which was used for the determination of PSSG in biological samples. Analysis of rat liver samples clearly demonstrated that GSH and GSSG co-precipitated with proteins similar to the range for PSSG in the sample. These data demonstrate that GSH and GSSG contamination must be accounted for when determining PSSG content in cellular/tissue preparations. A protocol for rinsing samples in order to remove the adventitious glutathione species is also demonstrated. Finally the fragmentation patterns for glutathione were determined by high resolution mass spectrometry and candidate ions for direct detection of PSSG on protein and protein fragments were identified.