Department of Microbial Proteomics, Institute of Microbiology; University of Greifswald, Felix-Hausdorff-Str. 8, 17489 Greifswald, Germany.
Department of Microbial Physiology and Molecular Biology, Institute of Microbiology; University of Greifswald, Felix-Hausdorff-Str. 8, 17489 Greifswald, Germany.
J Proteome Res. 2020 Apr 3;19(4):1435-1446. doi: 10.1021/acs.jproteome.9b00615. Epub 2020 Mar 23.
To understand bacterial reactions to environmental stress or infection-related processes, it is necessary to identify the involved proteins. In mass spectrometry-based proteomics, the method of choice for spectra-to-peptide-match is database search, but in recent times, spectral libraries have come into focus. Here, we built a mass spectral library from D39, reflecting 76% of the theoretical proteome of the organism. Besides the proteins themselves, posttranslational protein modifications especially reveal central hubs of regulation in bacterial pathogenesis. Here, for example, phosphorylation events are involved in the signal transduction and regulation of virulence. Although there have been major advances in phosphoproteomics, identification of this modification is still challenging. To enhance the number of phosphorylated peptides, which can be reproducibly detected, a comprehensive mass spectral library of the D39 phosphoproteome has been compiled in addition to the comprehensive total proteome mass spectral library. The phosphopeptide library was manually validated, and the data quality was additionally proven by analyses of synthetic phosphorylated peptides. In total, 128 phosphorylated proteins were revealed, of which many are involved in glycolysis, purine metabolism, protein biosynthesis, and virulence. The publicly available, thoroughly validated spectral libraries are an excellent resource to improve and speed up future investigations on the proteome and phosphoproteome of pneumococci.
为了了解细菌对环境压力或感染相关过程的反应，有必要鉴定涉及的蛋白质。在基于质谱的蛋白质组学中，用于谱图到肽匹配的方法是数据库搜索，但近年来，光谱库已成为焦点。在这里，我们构建了一个来自 D39 的质谱库，反映了该生物体理论蛋白质组的 76%。除了蛋白质本身，翻译后蛋白质修饰，特别是在细菌发病机制的调控中，揭示了中心枢纽。例如，磷酸化事件涉及信号转导和毒力的调节。尽管磷酸蛋白质组学已经取得了重大进展，但这种修饰的鉴定仍然具有挑战性。为了增加可重复性检测到的磷酸化肽的数量，除了全面的总蛋白质组质谱库外，还编译了 D39 磷酸蛋白质组的全面质谱库。磷酸肽库经过了手动验证，并且通过分析合成的磷酸化肽进一步证明了数据质量。总共鉴定出 128 个磷酸化蛋白，其中许多涉及糖酵解、嘌呤代谢、蛋白质生物合成和毒力。这些公共的、经过彻底验证的光谱库是一个极好的资源，可以改进和加快未来对肺炎球菌蛋白质组和磷酸蛋白质组的研究。
