Injury　of　p16　has　been　implicated　in　some　cancers.　In　this　paper,　we　focus　on　the　need　for　identification　of　peroxynitrite-dependent　nitration　sites　on　p16　with　HPLC-MS/MS　method.　Two　mono-nitrated　residues　Tyr129　and　Tyr44　were　detected　in　the　course　of　p16　modification　induced　by　peroxynitrite　at　relative　low　doses.　As　suggested　by　peptide　mapping　sequence　analysis,　Tyr44　was　more　liable　to　be　nitrated　by　ONOO-.　Study　on　the　chemical　environment　of　two　Tyr　residues　reveals　that　steric　hindrance　may　be　the　structural　determinant　for　the　nitration　sequence.　Through　technique　of　SDS-PAGE,　ONOO-　could　induce　p16　nitration,　even　strongly　damage　the　combination　of　p16　with　CDK4,　which　further　influence　p16＇s　activity.