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

摘要

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 HPO_(3) (80 Da) is negligible from the deprotonated phosphotyrosine peptides. The efficient H_(3)PO_(4) release is unexpected, given the structure of phosphotyrosine. Our study reveals that the abundant [M - H - 98]~(-) productions of pTyr-peptides are not a result of consecutive losses of HPO_(3) and H_(2)O 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 H_(3)PO_(4) 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 H_(3)PO_(4) 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.