Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry
Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry
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DOI：
10.1021/ac000703z
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发表时间：
2001-01-01
影响因子：
7.4
通讯作者：
McLafferty, FW
中科院分区：
文献类型：
--
作者：
Shi, SDH;Hemling, ME;McLafferty, FW
在用于解离多电荷肽和蛋白质离子的方法中，电子捕获解离（ECD）具有在高比例的氨基酸之间切割而不损失诸如糖基化和羧化的翻译后修饰的优点。在此，这种能力被成功地扩展到磷酸化，对于磷酸化，碰撞活化解离（CAD）可以引起H（3）PO（4）和HPO（3）的大量损失，如图所示，这些损失在ECD光谱中是最小的，这是测量磷酸化程度的优势。对于磷酸化肽，ECD和CAD光谱给出互补的骨架裂解，用于鉴定修饰位点。对于24-kDa异质磷蛋白，牛β-酪蛋白，活化离子ECD切割208个骨架键中的87个，其鉴定了Ser-15处的磷酸化位点，并且在Ser-17、-18、-19和-22和Thr-24中定位了另外三个，并且在四个其它位点中定位了最后一个：这是第一个直接的位点特异性表征的这种关键的翻译后修饰的蛋白质，而没有其先前的降解，如蛋白水解。
Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation, Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of H(3)PO(4) and HPO(3), As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine beta -casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr-24, and the last among four other sites: This is the first direct site-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis.