In vivo protein tyrosine nitration in Arabidopsis thaliana

Nitration of tyrosine (Y) residues of proteins is a low abundant post-translational modification that modulates protein function or fate in animal systems. However, very little is known about the in vivo prevalence of this modification and its corresponding targets in plants. Immunoprecipitation, based on an anti-3-nitroY antibody, was performed to pull-down potential in vivo targets of Y nitration in the Arabidopsis thaliana proteome. Further shotgun liquid chromatography–mass spectrometry (LC-MS/MS) proteomic analysis of the immunoprecipitated proteins allowed the identification of 127 proteins. Around 35% of them corresponded to homologues of proteins that have been previously reported to be Y nitrated in other non-plant organisms. Some of the putative in vivo Y-nitrated proteins were further confirmed by western blot with specific antibodies. Furthermore, MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analysis of protein spots, separated by two-dimensional electrophoresis from immunoprecipitated proteins, led to the identification of seven nitrated peptides corresponding to six different proteins. However, in vivo nitration sites among putative targets could not be identified by MS/MS. Nevertheless, an MS/MS spectrum with 3-aminoY318 instead of the expected 3-nitroY was found for cytosolic glyceraldehyde-3-phosphate dehydrogenase. Reduction of nitroY to aminoY during MS-based proteomic analysis together with the in vivo low abundance of these modifications made the identification of nitration sites difficult. In turn, in vitro nitration of methionine synthase, which was also found in the shotgun proteomic screening, allowed unequivocal identification of a nitration site at Y287.