Simultaneous measurement of protein oxidation and S-nitrosylation during preconditioning and ischemia/reperfusion injury with resin-assisted capture

RATIONALE: Redox modifications play an important role in many cellular processes, including cell death. Ischemic preconditioning (IPC) has been shown to involve redox signaling. Protein S-nitrosylation (SNO) is increased following myocardial IPC, and SNO is thought to provide cardioprotection, in part, by reducing cysteine oxidation during ischemia/reperfusion (IR) injury. OBJECTIVE: To test the hypothesis that SNO provides cardioprotection, in part, by shielding against cysteine oxidation following IR injury. METHODS AND RESULTS: We developed a new method to measure protein oxidation using resin-assisted capture (Ox-RAC), which is similar to the SNO-RAC method used in the quantification of SNO. Langendorff-perfused hearts were subjected to various perfusion protocols (control, IPC, IR, IPC-IR, IPC/reperfusion) and homogenized. Each sample was divided into 2 equal aliquots, and the SNO-RAC/Ox-RAC procedure was performed to simultaneously analyze SNO and oxidation. We identified 31 different SNO proteins with IPC, 27 of which showed increased SNO compared to baseline. Of the proteins that showed significantly increased SNO with IPC, 76% showed decreased oxidation or no oxidation following ischemia and early reperfusion (IPC-IR) at the same site when compared to IR alone; for non-SNO proteins, oxidation was reduced by only 50%. We further demonstrated that IPC-induced protein SNO is quickly reversible. CONCLUSIONS: These results support the hypothesis that IPC-induced protein SNO provides cardioprotection by shielding cysteine residues from reactive oxygen speciesg-induced oxidation during IR injury. Therefore, the level of protein SNO plays a critical role in IR injury, where ROS production is increased.