McCormack A L, Schieltz D M, Goode B, Yang S, Barnes G, Drubin D, Yates J R
Department of Molecular Biotechnology, University of Washington, Seattle 98195-7730, USA.
Anal Chem. 1997 Feb 15;69(4):767-76. doi: 10.1021/ac960799q.
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. The 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.
研究了一种通过微柱反相液相色谱电喷雾电离串联质谱法（LC/MS/MS）直接鉴定混合物中所含蛋白质的方法。在该方法中，蛋白质混合物用蛋白水解酶消化以产生大量肽段。然后将复杂的肽混合物与串联质谱仪在线分离，获取大量串联质谱图。接着使用串联质谱图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在摩尔量相差30倍时仍能容易地被鉴定出来，鉴定结果具有可重复性，并且混合物中的蛋白质能在低飞摩尔水平被鉴定。基于这些研究，已开发出用于对通过免疫亲和沉淀、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富集的蛋白质进行直接LC/MS/MS分析的方法。本文所述方法为直接鉴定混合物中的蛋白质提供了一种快速方法。
