基于LC-MS／MS策略建立十字花科黑腐病菌蛋白质表达谱的方法研究

摘要

【目的】建立十字花科黑腐病菌（Xanthomonas campestris pv．campestris ，Xcc ）的蛋白质组学研究平台，用于分离、鉴定该菌的致病相关蛋白质。【方法】Xcc 8004经过液体培养，分别提取胞内蛋白质和胞外蛋白质，对所有蛋白质进行液体酶解，通过强阳离子交换色谱预分离多肽混合物，预分离后的馏分采用 EASY-nLC 结合LTQ-Orbitrap 质谱鉴定蛋白质表达谱。【结果】建立了基于 bottom-up 策略的蛋白质组鉴定方法，采用第一维离子交换预分离和第二维反相色谱分离结合，实现了高通量鉴定蛋白质组表达谱的技术体系。通过 SEQUEST 检索，在胞内蛋白质样品中共鉴定蛋白质数目为1595个，胞外蛋白质样品共鉴定蛋白质数目为1241个。【结论】建立的 LC-MS/MS 方法适合用于十字花科黑腐病菌蛋白质组学的研究，为研究微生物植物相互作用奠定了方法与理论基础。%[Objective ]A proteomics technical platform of Xanthomonas campestris pv . campestris (Xcc )was established for the separation and identification of pathogenic bacteria associated proteins.[Methods]The intracellular proteins and extracellular proteins of Xcc 8004 strains were extracted after liquid cultured.After in-solution digestion of total proteins,the peptides mixtures were separated by strong cation exchange (SCX)column.The proteome pro-files were acquired by EASY-nLC combined LTQ-Orbitrap mass spectrometer.[Results]Based on bottom-up method,the proteins were analyzed by cation exchange column and nanoflow re-versed-phase liquid chromatography.The high proteomic profile identification was realized.The mass spectrometry data were searched with SEQUEST.As a result,a total of 1595 proteins were obtained from intracellular proteins and a total of 1241 proteins were obtained from extra-cellular proteins.[Conclusion]LC-MS/MS work-flow could be utilized successfully in the pro-teomics research of Xcc pathogenesis,which pro-vides the basis for studing the interaction of mi-crobe and plant.