Observation of phosphorylation site-specific dissociation of singly protonated phosphopeptides

In ultraviolet photodissociation of phosphopeptide ions with a basic residue (arginine, lysine, or histidine) at the N-terminus, intense a_n − 97 peaks were observed. These ions were formed by cleavage at phosphorylated residues only. For multiply phosphorylated peptides, this site-specific cleavage occurred at every phosphorylated residue. H/D exchange studies showed that a_n − 97 was formed by H₃PO₄ loss from a_n+1 radical cations. The site-specificity of phosphate loss observed here is in contrast to the nonspecific phosphate loss from b_n and y_n reported previously. Characteristics of the reaction and its potential utility for phosphopeptide analysis are discussed.