基于多维液相色谱质谱组合分析的痢疾杆菌蛋白质基因组学

Proteogenomics analysis of Shigella by combined multidimensional liquid chromatography tandem mass spectrometry

目的应用多维液相色谱质谱组合体系为基础的蛋白质组学方法对福氏痢疾杆菌基因组注释进行完善。方法痢疾杆菌福氏2a型301株(Sf2a301)的全菌蛋白经胰酶消化,二维液相色谱分离后进行MALDI-TOF/TOF和ESI-MS/MS组合鉴定,质谱数据分别应用MASCOT和SEQUEST软件检索基于Sf2a301全基因组构建的6个读码框数据库,完成对原基因组注释的验证和补充。结果研究表明多维液相色谱质谱组合体系能够增加鉴定蛋白的覆盖率,共鉴定Sf2a301的1231个蛋白编码基因产物,涵盖了COGs数据库22个功能分类组中的20个,包含306个功能未知的假定蛋白。发现了9个未注释的基因,得到RT-PCR和Northern blot的进一步验证。新基因大多数是重叠基因,包含3个嵌套基因。结论多维液相色谱质谱组合体系相对于单一的串联质谱技术能够更加有效验证、补充痢疾杆菌的基因组注释,更新后的基因组注释库为今后开展痢疾杆菌功能研究提供更多的靶点。

To apply proteomics method based on the combined multidimensional liquid chromatography mass spectrometry to the genome annotation of S.flexneri,bacterial proteins of S.flexneri 2astr.301（Sf2a301）were digested with trypsin,and peptides were separated by using two-dimensional liquid chromatography,and subsequently identified by using MALDITOF/TOF and ESI-MS/MS.Spectra data were searched respectively with MASCOT and SEQUEST against all possible sixframe translation database generated from whole genome sequences of S.flexneri to confirm and complement the genome annotation of Sf2a301.The research showed that proteome coverage could be greatly improved under the combination analysis of multi-dimensional liquid chromatography mass spectrometry.A total of 1 231 proteins of Sf2a301 were unambiguously identified in this research,involved in 20 groups of 22 functional groups in COGs.Meanwhile,306 hypothetical proteins were validated.Nine novel genes were discovered.Furthermore,novel genes were confirmed by RT-PCR or Northern blot experiment.Most of novel genes were overlapping genes,three even nested within the coding region of other known genes.Compared to any single tandem mass spectrometry,combined multidimensional liquid chromatography mass spectrometry can better validate and complement the genome annotation of S.flexneri.The updated database could provide more targets for those interested in Shigellato perform functional studies.