- 第3日 5月17日（木） 16:15～16:35 A会場（オービットホール）
Proteomic-scale approaches for quantifying irreversible cysteine redox post-translational modifications using parallel reaction monitoring mass spectrometry in myocardial ischemia / reperfusion
Redox post-translational modification (PTM) is an important defence against, and marker of, pathogenesis. Cysteine (Cys) is the most redox active amino acid and Cys-redox PTMs are considered either biologically reversible (e.g. S-glutathionylation) or irreversible (sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids), the latter of which ‘tags' proteins for degradation. Irreversible redox PTMs are considered the least abundant modifications under physiological conditions. We developed an enrichment method to examine these PTMs on a proteome-scale that employs electrostatic repulsion of Cys-SO2H/SO3H-containing peptides from cationic resins (‘negative' selection) followed by ‘positive' selection using hydrophilic interaction chromatography (HILIC). We identified >300 Cys-SO2H/SO3H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H2O2 (<100µM) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in mitochondrial fatty acid biosynthesis and the tricarboxylic acid cycle. A multiplexed quantitative analysis of both reversible and irreversible Cys PTMs identified redox targets in response to I/R and in the presence of a thiol-based antioxidant (N-2-mercaptopropionylglycine). Relative quantification of irreversibly oxidized Cys peptides was performed by parallel reaction monitoring (PRM-MS) to profile reversible/irreversible Cys PTMs in response to oxidants/antioxidants during I/R injury.