Identification of isomeric spin adducts of Leu–Tyr and Tyr–Leu free radicals using liquid chromatography–tandem mass spectrometry

ABSTRACTFree radical species are generally short‐lived due to their high reactivity and thus direct measurement and identification are often impossible. In this study we used a spin trap, 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO), to trap radical intermediates formed during the oxidation of isomeric dipeptides tyrosine–leucine (Tyr–Leu) and leucine–tyrosine (Leu–Tyr), induced by the hydroxyl radical. To investigate the influence of the amino acid position in the peptide chain on the oxidation and free radical generation, the spin adducts were characterized using LC‐MS and MSn. We detected carbon and oxygen DMPO adducts and adducts bearing two DMPO, which were analyzed by MSn. Both alkoxyl and peroxyl radicals were identified. Radical intermediates were localized in Tyr during oxidation of Tyr–Leu, while radicals were identified in Leu and Tyr during oxidation of Leu–Tyr. DMPO adducts of acyl radical species formed from cleavage of the peptide backbone, promoted by the alkoxyl radical in α carbon of the N‐terminal amino acid were observed. The results show that the amino acid position has an influence in the oxidation process, at least on small peptides, and that the α carbon of the N‐terminal amino acid is more vulnerable to the attack of the electrophilic hydroxyl radical. Copyright © 2011 John Wiley & Sons, Ltd.