枯草芽孢杆菌代谢组样品前处理方法的比较研究  

Comparative Study on Sample Pretreatment Methods for Metabolomics Analysis of Bacillus Subtilis

作　　者：王洪彬[1,2,3] 杨泓喆[1,2,3] 杨霁菡 王永帅[1,2,3] 路福平[1,2,3] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]天津科技大学工业发酵微生物教育部重点实验室,天津300457 [3]天津科技大学工业酶国家工程实验室,天津300457

出　　处：《分析化学》2015年第8期1169-1174,共6页Chinese Journal of Analytical Chemistry

基　　金：863计划项目(No.2013BAD10B01-5);国家自然科学基金项目(No.21306140)资助~~

摘　　要：本研究比较了不同淬灭剂（甲醇、60%甲醇、60%甲醇-0.9%Na Cl、60%甲醇-0.9%（NH4）2CO3和60%甲醇-0.9%Na Ac）条件下胞内物质核酸、蛋白质和代谢物的渗漏情况,发现60%甲醇-0.9%（NH4）2CO3和60%甲醇-0.9%Na Ac渗漏最少;比较了珠磨法、液氮研磨法、超声破碎3种细胞破碎方法条件下代谢物的提取效果,发现珠磨法提取效果最佳;比较了不同衍生时间（0.5~2.5 h）下代谢物相对丰度的变化,发现衍生时间达到2.0 h后,代谢物丰度不再有显著变化。最终确定了枯草芽孢杆菌代谢组样品最适前处理方法：预冷的60%甲醇-0.9%（NH4）2CO3淬灭,珠磨法破碎细胞,细胞破碎物用O-Bis[三甲基硅烷]三氟乙酸盐（MSTFA）衍生2 h。采用本方法从枯草芽孢杆菌中共鉴定到223种代谢物,包括大量氨基酸、有机酸和糖类物质。本研究为枯草芽孢杆菌的代谢组学研究提供了基础。An appropriate sample pretreatment method is crucial to the metabolomic analysis. Till now, many different sample pretreatment techniques for microbial metabolomies have been reported, but it is not clear which one is suitable for sample preparation for metabolomic analysis of Bacillus Subtilis. To improve the quality of metabolomic analysis of B. subtilis based on gas chromatography-mass spectrometry （ GC-MS）, in this study, the leakage of intracellular metabolites with five different quenching solvents was compared, and was found that 60% methanol/0.9% （NH4）2CO3 and 60% methanol/0.9% NaAC had the least leakage it The effects of different cell disruption methods including bead-milling, liquid nitrogen grounding and uhrasonication on the extraction of metabolites were compared. The bead-milling method exhibited the highest extraction efficiency. The effects of derivatization time （0. 5-2.5 h） on relative abundance of metabolite distribution were also investigated and it was found that 2 and 2.5 h were adequate for derivatization. The optimal sample pretreatment method for metabolomic analysis of B. subtilis was determined as follows： 60% methanol/0.9% （NH,）2CO3 as quencher, glass bead breaker used for cell disruption and the derivatization time of 2 h. With this method, 223 metabolites in B. subtilis, were identified by GC-MS, including a large number of amino acids, organic acids and carbohydrates. This study lays the foundation for the comprehensive Metabolomics analysis of Bacillus Subtilis.

关 键 词：代谢组学 枯草芽孢杆菌 气相色谱-质谱联用 前处理 

分 类 号：Q936[生物学—微生物学]