Analysis of peptides and proteins containing nitrotyrosine by matrix-assisted laser desorption/ionization mass spectrometry

Oxidative damage to proteins can occur under physiological conditions throu gh the action of reactive oxygen species, including those containing nitrog en such as peroxynitrite (ONO2-). Peroxynitrite has been shown in vitro to target tyrosine residues in proteins through free radical addition to produ ce S-nitrotyrosine. In this work, we show that mass spectral patterns assoc iated with 3-nitrotyrosine containing peptides allow identification of pept ides containing this modification. Matrix-assisted laser desorption/ionizat ion (MALDI) mass spectrometry was used to characterize a synthetic peptide AAFGY(m-NO2)AR and several peptides containing S-nitrotyrosine derived from bovine serum albumin treated with tetranitromethane. A unique series of io ns were found for these peptides in addition to the mass shift of +45 Da co rresponding to the addition of the nitro group. Specifically, two additiona l ions were observed at roughly equal abundance that correspond to the loss of one and two oxygens, and at lower abundances, two ions are seen that su ggest the formation of hydroxylamine and amine derivatives. These latter fo ur components appear to originate by laser-induced photochemical decomposit ion. MALDI-MS analysis of the synthetic peptide containing 3-nitrotyrosine revealed this same pattern. Post-source decay (PSD) MALDI-time-of-flight (T OF) and collisional activation using a prototype MALDI quadrupole TOF yield ed extensive fragmentation that allowed site-specific identification of S-n itrotyrosine. Conversion of peptides containing 3-nitrotyrosine to 3-aminot yrosine with Na2S2O4 yielded a single molecular ion by MALDI with an abunda nt sidechain loss under PSD conditions. These observations suggest that MAL DI can provide a selective method for the analysis and characterization of 3-nitrotyrosine-containing peptides. (C) 2001 American Society for Mass Spe ctrometry.