Peroxynitrite　(ONOO),　the　product　of　superoxide　(O2·　-)　and　nitric　oxide　(NO),　has　been　implicated　in　diabetes　and　diabetic　cardiovascular　complications.　Insulin,　a　mediator　of　these　pathological　processes,　has　an　important　role　to　maintain　human　blood　glucose　levels,　and　may　be　a　potential　target　during　accumulating　ONOO　is　formed　in　β-cells.　ONOO　can　inhibit　the　biological　activity　of　insulin　and　affect　its　receptor　binding　capacities　by　modifying　protein　tyrosine　residues.　Herein,　we　analyzed　the　dose-dependent　insulin　tyrosine　modification　indicated　by　spectral　changes.　Also,　the　nitrated　sites　of　insulin　were　identified　by　HPLC-MS　analysis.　The　results　show　that　an　infusion　of　ONOO　caused　a　maximum　observed　yield　of　2.7　3-nitrotyrosine　per　insulin　subunit　and　led　to　different　nitrated　species.　Moreover,　Try-B26　appeared　to　be　the　most　susceptible　residue　concerning　with　ONOO　mediated　nitration.　©　2007　IEEE.