Protein identification via ion-trap collision-induced dissociation and examination of low-mass product ions
Protein identification via ion-trap collision-induced dissociation and examination of low-mass product ions
复制标题
DOI：
10.1002/jms.1263
复制
发表时间：
2008-01-01
影响因子：
2.3
通讯作者：
McLuckey, Scott A.
中科院分区：
文献类型：
--
作者：
Bowers, Jeremiah J.;Liu, Jian;McLuckey, Scott A.
描述并评估了一种基于前体离子电荷态浓度的蛋白质鉴定全蛋白串联质谱方法，该方法通过离子/离子反应、离子阱碰撞激活、涉及产物离子的离子/离子质子转移反应以及窄m/z范围（高达m/z 2000）的质量分析。实验是用市售的电喷雾离子阱仪器进行的，该仪器经过修改以允许离子/离子反应。在假设质谱分析仪的分辨能力不足以根据同位素间距区分电荷状态的情况下，开发了反应条件和蛋白质数据库搜索方法。从细胞色素c、肌红蛋白、核糖核酸酶A和泛素的几种电荷状态中获得的离子被用来评估蛋白质鉴定方法，并开发了一个两步程序来数据库搜索以优化特异性。用模型蛋白开发的方法随后应用于酿酒酵母的全细胞裂解物。结果用三个先验未知蛋白的赋值举例说明，每个都是从裂解物分数中随机选择的。这三种蛋白质中的两种被分配给数据库中存在的物种，而一种与任何数据库条目都不匹配。质量测量和产物离子质量的结合表明，数据库中蛋白质的两个蛋氨酸残基可能被氧化。这些例子表明，这种有限的全蛋白表征方法可以提供基于完全酶消化的方法可能缺乏的见解。版权所有(c) 2007约翰威利父子有限公司
A whole-protein tandem mass spectrometry approach for protein identification based on precursor ion charge state concentration via ion/ion reactions, ion-trap collisional activation, ion/ion proton-transfer reactions involving the product ions, and mass analysis over a narrow m/z range (up to m/z 2000) is described and evaluated. The experiments were carried out with a commercially available electrospray ion-trap instrument that has been modified to allow for ion/ion reactions. Reaction conditions and the approach to searching protein databases were developed with the assumption that the resolving power of the mass analyzer is insufficient to distinguish charge states on the basis of the isotope spacings. Ions derived from several charge states of cytochrome c, myoglobin, ribonuclease A, and ubiquitin were used to evaluate the approach for protein identification and to develop a two-step procedure to database searching to optimize specificity. The approach developed with the model proteins was then applied to whole cell lysate fractions of Saccharomyces cerevisiae. The results are illustrated with examples of assignments made for three a priori unknown proteins, each selected randomly from a lysate fraction. Two of the three proteins were assigned to species present in the database, whereas one did not match well any database entry. The combination of the mass measurement and the product ion masses suggested the possibility for the oxidation of two methionine residues of a protein in the database. The examples show that this limited whole-protein characterization approach can provide insights that might otherwise be lacking with approaches based on complete enzymatic digestion. Copyright (c) 2007 John Wiley & Sons, Ltd.