title: Quantitation of protein S-glutathionylation by liquid chromatography-tandem mass spectrometry: correction for contaminating glutathione and glutathione disulfide.
abstract: Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein-glutathione mixed disulfide (PSSG) is commonly quantified by 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. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed for quantification of glutathione and glutathione disulfide (GSSG), which was used for the determination of PSSG in biological samples. Analysis of rat liver samples demonstrated that GSH and GSSG coprecipitated with proteins similar to the range for PSSG in the sample. The use of [(13)C2,(5)N]GSH and [(13)C4,(5)N2]GSSG validated these results and demonstrated that the release of GSH from PSSG did not occur during sample preparation and analysis. 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 to remove the adventitious glutathione species is demonstrated. The fragmentation patterns for glutathione were determined by high-resolution mass spectrometry, and candidate ions for detection of PSSG on protein and protein fragments were identified.
authors.affiliation: U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS), Grand Forks Human Nutrition Research Center, Grand Forks, ND 58203, USA. Electronic address: michael.bukowski@ars.usda.gov.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Bukowski
authors.affiliation: U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS), Grand Forks Human Nutrition Research Center, Grand Forks, ND 58203, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bucklin
authors.affiliation: U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS), Grand Forks Human Nutrition Research Center, Grand Forks, ND 58203, USA; Department of Chemistry, University of North Dakota, Grand Forks, ND 58203, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Picklo
mesh_terms: D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000032:analysis*; D019803:Glutathione Disulfide / Q000032:analysis*; D007553:Isotope Labeling; D008099:Liver / Q000378:metabolism; D008297:Male; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D005978:Glutathione; D019803:Glutathione Disulfide
keywords: Disulfide; Glutathione; Glutathionylation; LC–MS/MS; Protein