Fig. 5.

General protocol for the manual validation of MS/MS-based identification of nitrated peptides derived from the tyrosine nitration analyses presented in this study. Carbamoylation and deamidation should be explored as potential isobaric artifacts given that these particular modifications can occur in vivo in addition to the possibility of in vitro modification if urea was used for sample processing. Other important factors that should be considered include the size of the peptide (>7 amino acid residues) and identification of the unmodified form of the nitrated peptide given the substoichiometric nature of nitration. *, based on MS/MS analyses of nitrated peptide standards from this study, no modification-specific fragmentation was observed for nitration.