Insulin nitration mediated by peroxynitrite (ONOO(-)) has been implicated in diabetes and diabetic cardiovascular complications. In this study, we identified the nitration sites of porcine insulin by infusion of ONOO(-) and quantified its secondary structural change. Insulin was cleaved with V8 protease to six peptides (four of them contained each tyrosine residue), then analyzed by HPLC-MS and further confirmed the nitration sites by HPLC-MS/MS. At low accumulated doses of peroxynitrite, the main products were two different mono-nitrated insulin species at Tyr-A19 and Tyr-B26 with Tyr-A19 being predominant as shown by peptide mapping. Also, the content of α-helix structure of insulin reduced to 22.9 % and random-coil structure increased to 30.2 % (compare with native insulin of 41.7 % and 13.7 %, respectively) as determined by FTIR spectra.