Efficient Identification and Quantification of Peptides Containing Nitrotyrosine by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry after Derivatization

Protein nitration at tyrosine residues proceeds to form 3-nitrotyrosine; this product is today used as a biomarker of nitrative stress. We have developed an efficient method with which to identify nitrated peptides and to quantify protein nitration levels in different biological samples by a combination of the chemical derivatization of nitrotyrosine-containing peptides and mass spectrometry. Our strategy includes: 1) the protection of both N-terminal amines and epsilon-amines of lysine residues by acetylation with (13)C(0)/(13)C(4)- or D(0)/D(6)-acetic anhydride; 2) the reduction of nitrotyrosine to aminotyrosine with sodium hydrosulfite; and 3) the derivatization of aminotyrosine with 1-(6-methyl[D(0)/D(3)]nicotinoyloxy)succinimide. The utility of our method is demonstrated with nitrotyrosine-containing angiotensin II and bovine serum albumin as the model compounds.