Faculty of Chemistry, University of Wrocław, 50-383 Wrocław, Poland.
Biosci Rep. 2010 Dec;30(6):433-43. doi: 10.1042/BSR20090167.
Phosphorylation of proteins is an essential signalling mechanism in eukaryotic and prokaryotic cells. Although N-phosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in terms of products and mechanisms. In the present study, two MS fragmentation methods, ECD (electron-capture dissociation) and CID (collision-induced dissociation), were tested as tools for analysis of N-phosphorylation of three model peptides, RKRSRAE, RKRARKE and PLSRTLSVAAKK. The peptides were phosphorylated by reaction with monopotassium phosphoramidate. The results were confirmed by 1H NMR and 31P NMR studies. The ECD method was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main advantage is a significant reduction of the neutral losses related to the phosphoramidate moiety. Moreover, the results indicate that the ECD-MS may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities of the obtained lysine-phosphorylated peptides under various conditions were also tested.
蛋白质的磷酸化是真核和原核细胞中一种重要的信号转导机制。虽然 N-磷酸化碱性氨基酸在生物系统中的重要性已被广泛研究，但在产物和机制方面仍有待深入探讨。在本研究中，我们测试了两种 MS 碎裂方法，电子俘获解离（ECD）和碰撞诱导解离（CID），作为分析三种模型肽 RKRSRAE、RKRARKE 和 PLSRTLSVAAKK 的 N-磷酸化的工具。这些肽通过与单磷酸磷酰胺反应进行磷酸化。结果通过 1H NMR 和 31P NMR 研究得到了证实。ECD 方法对于不稳定的赖氨酸磷酸化肽中磷酸化位点的定位非常有用。它的主要优点是显著减少了与磷酰胺部分相关的中性损失。此外，结果表明 ECD-MS 可能有助于分析 N-磷酸化反应的区域选择性。还测试了在各种条件下获得的赖氨酸磷酸化肽的稳定性。
