Informatics and multiplexing of intact protein identification in bacteria and the archaea
Informatics and multiplexing of intact protein identification in bacteria and the archaea
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DOI：
10.1038/nbt1001-952
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发表时间：
2001-10-01
影响因子：
46.9
通讯作者：
Kelleher, NL
中科院分区：
文献类型：
--
作者：
Meng, FY;Cargile, BJ;Kelleher, NL
尽管在质谱仪中直接裂解蛋白质离子比详尽绘制1-3 kDa肽更有效，以完全表征从测序的基因组预测的一级结构，但这种方法的发展仍处于起步阶段。在这里，我们描述了一个统计模型（好到接近5%），表明该方法的数据库搜索特异性只需要四个碎片离子中的三个匹配（+/-0.1 Da），在5,000种蛋白质形式的数据库中有99.8%的正确概率。软件开发的蛋白质离子碎片数据的自动化处理和基于概率的检索整个蛋白质的说明，通过鉴定18古细菌和细菌蛋白质，同时质谱（MS）映射其整个一级结构。两个或三个蛋白质的解离一次这样的鉴定平行也被证明，沿着保留和准确定位的磷酸化丝氨酸残基通过片段化过程。这些概念和技术上的进步应有助于未来以更高通量的形式处理整个蛋白质，以更稳健地检测共翻译和翻译后修饰。
Although direct fragmentation of protein ions in a mass spectrometer is far more efficient than exhaustive mapping of 1-3 kDa peptides for complete characterization of primary structures predicted from sequenced genomes, the development of this approach is still in its infancy. Here we describe a statistical model (good to within similar to5%) that shows that the database search specificity of this method requires only three of four fragment ions to match (at +/-0.1 Da) for a 99.8% probability of being correct in a database of 5,000 protein forms. Software developed for automated processing of protein ion fragmentation data and for probability-based retrieval of whole proteins is illustrated by identification of 18 archaeal and bacterial proteins with simultaneous mass-spectro metric (MS) mapping of their entire primary structures. Dissociation of two or three proteins at once for such identifications in parallel is also demonstrated, along with retention and exact localization of a phosphorylated serine residue through the fragmentation process. These conceptual and technical advances should assist future processing of whole proteins in a higher throughput format for more robust detection of co- and post-translational modifications.