Increased O(2)(*-) and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In complex II, oxidative impairment and enhanced tyrosine nitration of the 70 kDa FAD-binding protein occur in the post-ischemic myocardium and are thought to be mediated by peroxynitrite (OONO(-)) in vivo [Chen, Y.-R., et al. (2008) J. Biol. Chem. 283, 27991-28003]. To gain deeper insights into the redox protein thiols involved in OONO(-)-mediated oxidative post-translational modifications relevant in myocardial infarction, we subjected isolated myocardial complex II to in vitro protein nitration with OONO(-). This resulted in site-specific nitration at the 70 kDa polypeptide and impairment of complex II-derived electron transfer activity. Under reducing conditions, the gel band of the 70 kDa polypeptide was subjected to in-gel trypsin/chymotrypsin digestion and then LC-MS/MS analysis. Nitration of Y(56) and Y(142) was previously reported. Further analysis revealed that C(267), C(476), and C(537) are involved in OONO(-)-mediated S-sulfonation. To identify the disulfide formation mediated by OONO(-), nitrated complex II was alkylated with iodoacetamide. In-gel proteolytic digestion and LC-MS/MS analysis were conducted under nonreducing conditions. The MS/MS data were examined with MassMatrix, indicating that three cysteine pairs, C(306)-C(312), C(439)-C(444), and C(288)-C(575), were involved in OONO(-)-mediated disulfide formation. Immuno-spin trapping with an anti-DMPO antibody and subsequent MS was used to define oxidative modification with protein radical formation. An OONO(-)-dependent DMPO adduct was detected, and further LC-MS/MS analysis indicated C(288) and C(655) were involved in DMPO binding. These results offered a complete profile of OONO(-)-mediated oxidative modifications that may be relevant in the disease model of myocardial infarction.