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 e-amines of lysine residues by acetylation with 13C0/13C4– or D0/D6–acetic anhydride; 2) the reduction of nitrotyrosine to aminotyrosine with sodium hydrosulfite; and 3) the derivatization of aminotyrosine with 1-(6-methyl[D0/D3]nicotinoyloxy)succinimide. The utility of our method is demonstrated with nitrotyrosine-containing angiotensin II and bovine serum albumin as the model compounds.