Strategy for absolute quantification of proteins: CH3Hg+labeling integrated molecular and elemental mass spectrometry
Strategy for absolute quantification of proteins: CH3Hg+labeling integrated molecular and elemental mass spectrometry
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DOI：
10.1039/b902241d
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发表时间：
2009-08
影响因子：
3.4
通讯作者：
Yi-Chao Guo;Ming Xu;Limin Yang;Qiuquan Wang
中科院分区：
文献类型：
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作者：
Yi-Chao Guo;Ming Xu;Limin Yang;Qiuquan Wang
目前，分子质谱仪是相对定量的首选方法，但不适合蛋白质的“绝对”定量。在这篇文章中，我们展示了一种通过CH3Hg+标记和分子与元素质谱学的综合应用来进行蛋白质绝对定量分析的概念证明。ESI-MS证实，在单烷基汞化合物中，CH3Hg+的尺寸最小，并且与蛋白质中的巯基(-SH)发生特异的共价相互作用，当暴露于所有-SH时，形成CH3Hg+：-SH=1：1的简单络合物。根据已知的每种蛋白质的-SH数，用电感耦合等离子体质谱(ICMS)测定蛋白质的绝对浓度，可以简单地用CH3HgCl2作为外标。以分子中各种二硫键数目增加的牛胰腺核糖核酸酶A、溶菌酶和胰岛素为模型蛋白时，其相应的绝对检测限(3σ)分别为0.6pmol、1.2pmol和0.4pmo.这些特性有望在不久的将来为蛋白质的绝对定量，特别是特定生物标志物的测定提供一种替代方法。
Currently, molecular mass spectrometry is preferred by many for relative quantification but is not appropriate for “absolute” quantification of proteins. In this article we demonstrate a proof of concept for the absolute quantitative analysis of proteinsvia CH3Hg+labeling and integrated application of molecular and elemental mass spectrometry. The smallest size of CH3Hg+ among monoalkyl mercurials and the specific and covalent interaction with sulfhydryl (–SH) in proteins results in forming a simple complex of CH3Hg+:–SH = 1:1 when all –SH are exposed, as confirmed by ESI-MS. Based on the known number of –SH per protein, the absolute protein concentration can be obtained viaHg determination using ICP-MS, in which CH3HgCl could be simply used as an external standard. When bovine pancreatic ribonuclease A, lysozyme and insulin, which have an increasing number of various disulfide linkages in their molecules, were taken as model proteins, their corresponding absolute detection limits (3σ) reached 0.6, 1.2 and 0.4 pmol, respectively. These characteristics may be expected to provide an alternative approach for absolute protein quantification, especially specific biomarker determination, in the near future.