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

Abstract

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](-) product ions 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.

Fragmentations of [M − H]− anions of peptides containing tyrosine sulfate. Does the sulfate group rearrange? A joint experimental and theoretical study

“…This loss of SO 3 is analogous to the loss of HOPO 2 from the [M À H] À anions of peptides containing Tyr(PO 3 H 2 ). [3,4] Scheme 2. Fig.…”

“…9. There is no particular fragmentation that identifies a corresponding and competitive sulfate rearrangement to a C-terminal CO 2 À ion in this system, but that does not exclude the possibility that such a process forms HOSO 3 À , at least in part.…”

“…The reaction coordinate diagram for the formation of HOSO 3 À and [(M À H) À H 2 SO 4 ] À following rearrangement of sulfate from Tyr to the Ser side chain is shown in Fig. 10.…”

“…In contrast, the negative ion cleavages of tyrosine, serine, and threonine (Tyr, Ser and Thr) phosphates [3,4] are complex, involving a variety of S N i(P) rearrangements often followed by internal cyclization/cleavage reactions . [4] The phosphate group of Tyr can transfer to the Ser and Thr residues, and also to a carboxylate (or amide) moeity.…”

Analysis of creatine phosphate disodium salt by ESI-MS/MS: A seven-centered rearrangement mechanism in gas phase

“…Mass spectrometry (MS), especially tandem mass spectrometry (MS/MS), is an important physicochemical method for the identification of trace compounds in the organism, as only low levels of sample are required and it is rapid and sensitive. Recently, the application of electrospray ionization mass spectrometry (ESI-MS) has been reported to analyze the phosphoryl molecules [4][5][6][7][8][9][10][11]. Interestingly, several phosphoryl group (P O) participated rearrangement reactions were reported in gas phase, such as phosphoTyr to C-terminal CO 2 PO 3 H 2 rearrangement [7], the phosphoryl rearrangement of P N to P O bonds [8,9], methoxyl group migration [10], and carbonyl oxygen migration [11].…”