Submitted to: American Society for Mass Spectrometry
Publication Type: Abstract Only
Publication Acceptance Date: 3/4/2015
Publication Date: 6/15/2014
Citation: Bukowski, M.R., Picklo, M.J. 2014. Measuring protein-bound glutathioine (PSSG): Critical correction for cytosolic glutathione species [abstract]. American Society for Mass Spectrometry. p. 58:MP089.
Technical Abstract: Introduction: Protein glutathionylation is gaining recognition as an important posttranslational protein modification. The common first step in measuring protein glutathionylation is the denaturation and precipitation of protein away from soluble, millimolar quantities of glutathione (GSH) and glutathione disulfide (GSSG). We demonstrate that contamination of precipitated GSH and GSSG, while only >1% of the total GSH pool, is a significant contributor to observed glutathionylation levels and that extensive rinsing of protein pellets and use of thiol alkylators is not sufficient to eliminate this contamination. Further, we demonstrate an LCMS-based method which controls for artifactual contamination and allows for the measurement of PSSG at the picomole/mg protein level, a level 104 times lower than GSH concentration in the cytosol. Method: Rat liver samples were homogenized in 5% sulfosalicylic acid (SSA) , the supernatant was removed and analyzed for free glutathione species. Pellets were rinsed, re-suspended in pH 7.4 buffer, and treated with the thiol alkylator N-ethyl maleimide (NEM) for 30 minutes at room temperature. Protein was reprecipitated through addition of acetonitrile, followed by sequential washing with acetonitrile to eliminate excess NEM. Pellets were resuspended in pH 9 buffer and split into control and experimental groups. The reducing agent tris(2-carboxyethyl) phosphine was added to experimental samples at room temperature for 40 minutes. Both groups received internal standard before the final protein precipitation with 5% SSA. Samples were analyzed by LC-MS, using characteristic MRM transitions to quantify GSH and GSSG. Results: Analysis of ten separate rat liver samples indicated that GSH and GSSG co-precipitate in the range of picomoles per mg protein. This is a critical problem with current literature in which the values for these species are unknown and assumed to be small. We demonstrate that corrections for the contaminating cytosolic GSH and GSSG are necessary for the quantification of PSSG. Without measurement and correction for adventitious GSH and GSSG the values for PSSG are reported 30 to 200 times higher than actually present. We also demonstrate that these contaminating glutathione species persist despite attempts to eliminate them, including pellet rinses and treatment with thiol-blocking agents. Novel Aspect: The contribution of contaminating glutathione species to PSSG measurement is quantified and corrected, a problem unaddressed in current literature.