A new approach for detecting C-terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization
A new approach for detecting C-terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization
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DOI：
10.1002/pmic.200900267
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发表时间：
2009-08-01
期刊：
影响因子：
3.4
通讯作者：
Tsunasawa, Susumu
中科院分区：
文献类型：
--
作者：
Kuyama, Hiroki;Nakajima, Chihiro;Tsunasawa, Susumu
我们描述了一种质谱方法，用于区分肽和蛋白质的 C 端羧基的游离形式和修饰形式，并结合化学方法分离 C 端肽和 C 端羧基的位点特异性衍生化。该方法可以最有利地用于区分具有游离(COOH)和酰胺(CONH(2))形式的C端羧基的肽，通过选择性地将游离羧基转化为甲酰胺(CONHCH(3))，将它们的质量差从1增加到14Da。该方法已被证明适用于含有天冬氨酸和谷氨酸的肽，因为根据恶唑酮化学，除 C 末端羧基外，所有羧基均对衍生化呈惰性。通过比较从肾上腺髓质素、降钙素和 BSA 的混合物中获得的加工肽的峰，说明了该方法的效率。在混合物的这些成分中，只有 BSA 的 C 端肽在处理后表现出 13 Da 的质量位移，最终明确地验证了肾上腺髓质素和降钙素的 C 端酰胺结构。还讨论了扩展该方法用于 C 末端 PTM 分析的可能性。
We describe a mass spectrometric method for distinguishing between free and modified forms of the C-terminal carboxyl group of peptides and proteins, in combination with chemical approaches for the isolation of C-terminal peptides and site-specific derivatization of the C-terminal carboxyl group. This method could most advantageously be exploited to discriminate between peptides having C-terminal carboxyl groups in the free (COOH) and amide (CONH(2)) forms by increasing their mass difference from 1 to 14 Da by selectively converting the free carboxyl group into methylamide (CONHCH(3)). This method has been proven to be applicable to peptides containing aspartic and glutamic acids, because all the carboxyl groups except the C-terminal one are inert to derivatization, according to oxazolone chemistry. The efficiency of the method is illustrated by a comparison of the peaks of processed peptides obtained from a mixture of adrenomedullin, calcitonin, and BSA. Among these components of the mixture, only the C-terminal peptide of BSA exhibited the mass shift of 13 Da upon treatment, eventually unambiguously validating the C-terminal amide structures of adrenomedullin and calcitonin. The possibility of extending this method for the analysis of C-terminal PTMs is also discussed.