Gas-Phase Intramolecular Phosphate Shift in Phosphotyrosine-Containing Peptide Monoanions

Phosphotyrosine-containing peptide monoanions [M - H](-) exhibit extensive neutral loss of phosphoric acid (98 Da) upon quadrupole time-of-flight and ion-trap collision-induced dissociation (CID). In contrast, a neutral loss of metaphosphoric acid HPO3 (80 Da) is negligible from the deprotonated phosphotyrosine peptides. The efficient H3PO4 release is unexpected, given the structure of phosphotyrosine. Our study reveals that the abundant [M - H - 98](-) product ions of pTyr-peptides are not a result of consecutive losses of HPO3 and H2O but, rather, are induced by an intramolecular interaction of the phosphotyrosine phosphate with deprotonated peptide functions such as hydroxyl, carboxyl, and to a small extent, amide. As a result, an internal phosphotyrosine phosphate shift occurs, and the obtained phosphorylated functionalities undergo elimination of H3PO4 to give rise to the [M - H - 98](-) fragments. The mechanism proposed for the phosphoric acid neutral loss is based on extensive CID studies of Ala-substituted model phosphorylated peptides and oxygen-18 labeling. The proposed mechanistic pathway explains the fact that the pTyr phosphate transfer and the subsequent H3PO4 neutral loss are not observed for multiply charged anions of pTyr-peptides. Monoanions of pSer-containing peptides undergo the intramolecular phosphate shift as well, although its efficiency is much lower compared to the aromatic phosphorylation sites. These observations facilitate correct identification of pSer-, pThr-, and pTyr-peptides in CID studies. This work demonstrates that the established phosphate-specific neutral loss fragmentation rules of protonated pTyr-peptides cannot be applied to the CID spectra of their [M - H](-) ions.