Investigation of tyrosine nitration in proteins by mass spectrometry

In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. Th e aim of this study was to evaluate possibilities for sit e-specific detect ion of tyrosine nitration by mass spectrometry. Angiotensin II and bovine s erum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated (i) analysis of single pepti des and protein digests by matrix-assisted laser desorption/ionization (MAL DI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionizat ion (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning wi th subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragm entation involving the nitro group, in contrast to ESI-MS, where no fragmen tation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment wit h TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by We stern blotting with an antibody against nitrotyrosine and the sites for nit ration were investigated by peptide mass mapping after in-gel digestion. Di rect mass mapping by ESI revealed that two peptides were nitrated. Precurso r ion scanning for the immonium ion for nitrotyrosine revealed two addition al partially nitrated peptides. Based on the studies with the two model com pounds, we suggest that the investigation of in ai oo nitration of tyrosine and identification of nitrated peptides might be performed by precursor io n scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS /MS for identification of the specific nitration sites. copyright (C) 2001 John Wiley & Sons, Ltd.