title: Simultaneous measurement of protein oxidation and S-nitrosylation during preconditioning and ischemia/reperfusion injury with resin-assisted capture.
abstract: BACKGROUND
  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.
  
  
  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 species-induced oxidation during IR injury. Therefore, the level of protein SNO plays a critical role in IR injury, where ROS production is increased.
authors.affiliation: Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21257, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Kohr
authors.forename: Junhui
authors.initials: J
authors.lastname: Sun
authors.forename: Angel
authors.initials: A
authors.lastname: Aponte
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.forename: Marjan
authors.initials: M
authors.lastname: Gucek
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Murphy
authors.forename: Charles
authors.initials: C
authors.lastname: Steenbergen
mesh_terms: D000818:Animals; D003545:Cysteine / Q000378:metabolism; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D008297:Male; D051379:Mice; D023421:Models, Animal; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000517:prevention & control; D009206:Myocardium / Q000378:metabolism*; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism*; D012117:Resins, Synthetic / Q000378:metabolism*; D026403:S-Nitrosothiols / Q000378:metabolism*
chemical_list: D011506:Proteins; D012117:Resins, Synthetic; D026403:S-Nitrosothiols; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D003545:Cysteine