Clokie Martha R J, Thalassinos Konstantinos, Boulanger Pascale, Slade Susan E, Stoilova-McPhie Svetla, Cane Matt, Scrivens James H, Mann Nicholas H
Department of Infection, Immunity and Inflammation, Maurice Shock Medical Sciences Building, University of Leicester, Leicester LE1 9HN, UK.
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Microbiology (Reading). 2008 Jun;154(Pt 6):1775-1782. doi: 10.1099/mic.0.2007/016261-0.
In this study, an MS-based proteomics approach to characterizing the virion structural proteins of the novel marine 'photosynthetic' phage S-PM2 is presented. The virus infects ecologically important cyanobacteria of the genus Synechococcus that make a substantial contribution to primary production in the oceans. The S-PM2 genome encodes 236 ORFs, some of which exhibit similarity to known phage virion structural proteins, but the majority (54%) show no detectable homology to known proteins from other organisms. Using public and in-house bioinformatics tools the proteome of S-PM2 was predicted and a database compatible with MS-based search engines was constructed. S-PM2 virion proteins were resolved by SDS-PAGE, excised, tryptically digested and analysed by LC-ESI-MS/MS. The resulting MS data were searched against the database. A parallel control study was undertaken on the well-characterized coliphage T4 in order to assess the sensitivity and efficiency of this approach. In total, 11 of the 15 S-PM2 proteins, predicted to be virion proteins by bioinformatics approaches, were confirmed as such, together with the identification of a further 12 novel structural proteins. In the case of T4, 24 of the 39 known virion structural proteins were identified, including the major tail-fibre proteins. This approach has wide-ranging applicability and can be applied to any novel organism whose genome encodes ORFs with few detectable homologies in the public databases.
在本研究中，我们提出了一种基于质谱的蛋白质组学方法，用于表征新型海洋“光合”噬菌体S-PM2的病毒粒子结构蛋白。该病毒感染对海洋初级生产有重大贡献的生态重要蓝藻属的聚球藻。S-PM2基因组编码236个开放阅读框（ORF），其中一些与已知噬菌体病毒粒子结构蛋白具有相似性，但大多数（54%）与其他生物体的已知蛋白没有可检测到的同源性。使用公共和内部生物信息学工具预测了S-PM2的蛋白质组，并构建了与基于质谱的搜索引擎兼容的数据库。通过SDS-PAGE分离S-PM2病毒粒子蛋白，切下条带，用胰蛋白酶消化并通过LC-ESI-MS/MS进行分析。将所得的质谱数据与该数据库进行比对搜索。对特征明确的大肠杆菌噬菌体T4进行了平行对照研究，以评估该方法的灵敏度和效率。通过生物信息学方法预测为病毒粒子蛋白的15种S-PM2蛋白中，共有11种得到了确认，同时还鉴定出另外12种新型结构蛋白。对于T4噬菌体，鉴定出了39种已知病毒粒子结构蛋白中的24种，包括主要尾丝蛋白。这种方法具有广泛的适用性，可应用于其基因组编码的开放阅读框在公共数据库中几乎没有可检测到的同源性的任何新型生物体。
