Gas-Phase Intramolecular Phosphate Shift in Phosphotyrosine-Containing Peptide Monoanions
Gas-Phase Intramolecular Phosphate Shift in Phosphotyrosine-Containing Peptide Monoanions
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DOI：
10.1021/ac900244e
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发表时间：
2009-06-01
影响因子：
7.4
通讯作者：
Lehmann, Wolf D.
中科院分区：
文献类型：
--
作者：
Edelson-Averbukh, Marina;Shevchenko, Andrej;Lehmann, Wolf D.
含磷酸酪氨酸的肽单阴离子[M-H]（-）在四极杆飞行时间和离子阱碰撞诱导解离（CID）后表现出广泛的磷酸中性损失（98 Da）。相比之下，偏磷酸HPO 3（80 Da）的中性损失是可以忽略不计的去质子化的磷酸酪氨酸肽。考虑到磷酸酪氨酸的结构，有效的H3 PO 4释放是出乎意料的。我们的研究表明，pTyr-肽的丰富的[M-H- 98]（-）产物离子不是HPO 3和H2O连续损失的结果，而是由磷酸酪氨酸磷酸与去质子化肽功能（如羟基、羧基和少量酰胺）的分子内相互作用诱导的。结果，发生内部磷酸酪氨酸磷酸移位，并且所获得的磷酸化官能团经历H3 PO 4的消除以产生[M-H- 98]（-）片段。提出的磷酸中性损失的机制是基于广泛的CID研究丙氨酸取代的模型磷酸化肽和氧-18标记。所提出的机理途径解释了这样一个事实，即pTyr磷酸转移和随后的H3 PO 4中性损失没有观察到pTyr-肽的多电荷阴离子。含pSer的肽的单阴离子也经历分子内磷酸盐移位，尽管其效率比芳香族磷酸化位点低得多。这些观察结果有助于在CID研究中正确鉴定pSer-、pThr-和pTyr-肽。这项工作表明，质子化pTyr-肽的磷酸盐特异性中性损失碎片化规则不能应用于其[M-H]（-）离子的CID光谱。
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.