A simple test to detect hydrogen/deuterium scrambling during gas-phase peptide fragmentation
A simple test to detect hydrogen/deuterium scrambling during gas-phase peptide fragmentation
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DOI：
10.1021/ac800645f
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发表时间：
2008-09-01
影响因子：
7.4
通讯作者：
Coales, Stephen J.
中科院分区：
文献类型：
--
作者：
Hamuro, Yoshitomo;Tomasso, Justine C.;Coales, Stephen J.
酰胺氢/氘（H/D）交换与质谱联用已成为研究蛋白质动力学的有力工具。在交换反应和质量分析之间添加蛋白水解步骤可用于定位氘的位置并提高总体分辨率。如果肽上的可交换氢和氘不发生混乱，则可以通过在气相中裂解消化的肽来进一步增强分辨率。虽然一些实验室报告说，成功地定位氘的气相碎裂，其他人描述的总混乱。在这里，我们提出了一个简单的方法来检测是否存在的混乱使用市售的小肽，神经降压素（9-13; RPYIL）。这个五肽上的所有可交换氢首先通过将其溶解在氧化氘中而被氘化。将氘代肽加载到反相柱上，然后用大量冷的酸性缓冲水溶液洗涤。这种洗涤将末端和侧链上的所有氘交换回氢。现在只有三个氘连接在五肽上，Y、1和L的酰胺氮上各一个。用95%酸性乙腈从柱上洗脱部分氘代肽后，碰撞诱导解离（CID）产生一系列B离子，这些离子通过质谱仪进行分析。在不加扰的情况下，在B（2）离子中不应观察到氘，因为R和P都没有酰胺氢。另一方面，在混乱的情况下，b2应该携带母体五肽的大约一半的氘。从理论上讲，完全混乱应该在所有可交换的氢中平均分配氘。神经降压素（9-13）的b2部分有6个可交换的氢，而神经降压素（9-13）的+1电荷状态有12个可交换的氢。我们证明了CID导致氢和氘的LCQ（离子阱机）的完全混乱。
Amide hydrogen/deuterium (H/D) exchange coupled with mass spectrometry has become a powerful tool to study protein dynamics. Addition of a proteolysis step between the exchange reaction and mass analysis can be used to localize the positions of deuterium and improve overall resolution. The resolution can be further enhanced by the fragmentation of digested peptides in the gas phase if scrambling of exchangeable hydrogens and deuteriums on the peptides does not occur. Although some laboratories reported successful localization of deuteriums by gas-phase fragmentations, others described total scrambling. Here we propose a simple method to detect the presence or absence of scrambling using a commercially available small peptide, neurotensin (9-13; RPYIL). All exchangeable hydrogens on this pentapeptide are first deuterated by dissolving it in deuterium oxide. The deuterated peptide is loaded onto a reversed-phase column, and then washed with copious amounts of cold acidic aqueous buffer. This washing exchanges all deuteriums on both the terminals and the side chains back to hydrogens. Now only three deuteriums are attached on the pentapeptide, one on each of the amide nitrogens of Y, 1, and L After the partially deuterated peptide is eluted from the column with 95% acidic acetonitrile, collision-induced dissociation (CID) generates a series of b ions, which are analyzed by mass spectrometer. In the absence of scrambling, no deuterium should be observed in the b(2) ion, as neither R nor P have amide hydrogens. On the other hand, in the event of scrambling, b2 should carry about half of the deuteriums of the parent pentapeptide. In theory, complete scrambling should distribute deuteriums equally among all of the exchangeable hydrogens. The b2 portion of neurotensin (9-13) has 6 exchangeable hydrogens, whereas the +1 charge state of neurotensin (9-13) has 12 exchangeable hydrogens. We demonstrated that CID caused complete scrambling of hydrogens and deuteriums with an LCQ (a ion trap machine).