Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level
Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level
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DOI：
10.1021/ac960799q
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发表时间：
1997-02-15
影响因子：
7.4
通讯作者：
Yates, JR
中科院分区：
文献类型：
--
作者：
McCormack, AL;Schieltz, DM;Yates, JR
研究了微柱反相液相色谱电喷雾电离串联质谱(LC/MS/MS)直接鉴定混合物中蛋白质的方法。在此方法中，用蛋白水解酶消化蛋白质混合物以产生大量肽。然后用串联质谱仪在线分离饼状复杂肽混合物，获取大量串联质谱，然后使用串联质谱搜索蛋白质数据库以鉴定存在的蛋白质。标准蛋白质混合物的结果表明，简单混合物中存在的蛋白质可以很容易地以 30 倍摩尔量差异进行鉴定，鉴定具有可重复性，并且可以在低飞摩尔水平下鉴定混合物中的蛋白质。基于这些研究，我们开发了用于直接 LC/MS/MS 分析通过免疫亲和沉淀富集的蛋白质、与蛋白质-蛋白质融合产物的特异性相互作用以及与大分子复合物的特异性相互作用的方法。本文中描述的方法为直接鉴定混合物中的蛋白质提供了一种快速方法。
A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of, proteins is digested with a proteolytic enzyme to produce a large collection of peptides. ?Pie complex peptide mixture is then separated on-line with a tandem mass spectrometer, acquiring large numbers of tandem mass spectra, The tandem mass spectra are then used to search a protein database to identify the proteins present. Results from standard protein mixtures show that proteins present in simple mixtures can be readily identified with a 30-fold difference in molar quantity, that the identifications are reproducible, and that proteins within the mixture can be identified at low femtomole levels. Based on these studies, methodology has been developed for direct LC/MS/MS analysis of proteins enriched by immunoaffinity precipitation, specific interaction with a protein-protein fusion product, and specific interaction with a macromolecular complex, The approach described in this article provides a rapid method for the direct identification of proteins in mixtures.