短芽孢杆菌代谢物组测定中抽提方法的比较分析 - yzxzcom.doc

短短芽孢杆菌代谢物组测定中灭活方式的优化 陈梅春，刘波*，朱育菁，陈峥，史怀，刘国红，潘志针 福建省农业科学院农业生物资源研究所，福建 福州350003 摘 要：微生物代谢组学在发酵控制分析、代谢物控制分析及其它组学联用进行菌种改良等方面得到了应用，其定性和定量分析细胞内外中所有低分子量代谢产物。胞内代谢物变化非常迅速，因此快速冻结代谢反应，使代谢物失活是非常重要的，必须有效率高和可靠的灭活方式，减少代谢物的流失。本研究以短短芽孢杆菌JK-2为研究对象，以LC-MS测定为评价标准，考察了五种灭活方式对胞内代谢物保留程度。结果表明，冷甲醇/水(含HEPES)灭活方法能够保留的代谢物最多，效率最高及重现性好，在LC-MS正离子模式下，能够提取出三千多种胞内代谢物。同时也考察了样品在正负模式下测试情况,结果表明正离子模式下能够检测到的代谢物的数目远远超过负离子模式下检测的数目。 关键词：短短芽胞杆菌、代谢组学、灭活、胞内代谢物、正负离子模式 中图分类号： The optimizing quenching solution for metabolite analysis of Brevibacillus brevis CHEN Mei-chun, LIU Bo, ZHUYu-jin, CHEN Zhen, SHI Huai, LIU Guo-hong, PAN Zhi-zhen Agricultural Bioresources Research Institute，Fujian Academy of Agricultural Sciences，Fuzhou，350003, China Abstract: Microbial metabolomics has been widely applied in the control of fermentation process, metabolites analysis and upgrading strains combined with other omics. Microbial metabolomics refers to the qualitative and quantitative analysis of the complete set of all low molecular weight metabolites present in and around the cells. For growing microorganisms, the turnover of intracellular metabolites can be extremely fast. Consequently, instantaneous fixation of the metabolism during sampling is essential. Therefore, the development of high efficiency and reliable quenching protocol is required to prevent significant leakage of intracellular metabolites. In this paper, five different quenching solutions are promoted to assess the leakage of intracellular metabolites based on LC-MS using the cold methanol extraction method. The results indicated that the cold-methanol/water (V:V = 3:2) containing the buffer agent HEPES quenching method for Brevibacillus brevis cell cultures is the highest efficiency and reproducible quenching methods, and more than 3000 intracellular metabolites can be detected under the positive ion ESI mode. The influence of the positive and negative ions ESI modes are also investigated which showed that the former can detect more metabolites than the later