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　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.