大肠杆菌胞内代谢物组学分析样品前处理条件优化

Optimization of Sample Preparation Method for Intracellular Metabolites Metabonomics Analysis of Escherichia Coli

建立了适用于大肠杆菌胞内代谢物组学分析的样品前处理条件。大肠杆菌菌体细胞采用冷冻盐水(0.85%NaCl,-80℃预冷15 min)进行猝灭,猝灭后的菌体细胞经过真空冷冻干燥,再通过液氮冷冻研磨结合超声裂解的方式通透细胞膜,最后采用冷甲醇-水(MeOH-H 2O,1∶1,V/V,4℃)提取胞内代谢物。分别采用流式细胞术检测和OD值回收率从单细胞水平和整体水平评价猝灭溶剂对大肠杆菌细胞膜损伤影响。结果表明,采用冷冻盐水猝灭大肠杆菌细胞损伤率小于5%。采用液相色谱-飞行时间质谱(T 3和Hilic 2种色谱分离结合ESI±扫描模式)检测结果中低碰撞能量提取出的峰数量和总离子强度评价了3种细胞通透方式和4种提取溶剂组合的胞内代谢物提取效果。结果表明,冷冻研磨结合超声裂解通透细胞膜、冷甲醇-水提取代谢物测得的代谢物数量最多(≥10^5),并且总离子强度在最优范围(10^6~10^7)内。本研究采用冷冻干燥、冷冻研磨和超声萃取相结合的代谢物提取方式,有效促进了细胞膜裂解,提高了代谢物提取效率。本研究建立的前处理条件适用于大肠杆菌胞内代谢物组学分析。

A sample preparation method was developed to simulate the process of intracellular metabolites metabonomics analysis of Escherichia coli.The Escherichia coli cell was firstly quenched with cold sodium chloride solution(0.85%,precooled at -80℃for 15 min).The quenched bacterial cell was treated by using the technique of vacuum freeze-drying and liquid nitrogen freezing combined with ultrasonic processing to increase cell membrane penetrability.Finally,a cold aqueous solution of methanol(MeOH∶H2O,1∶1,V/V,4℃)was used as extraction solvent to extract metabolites.In the present research,flow cytometry and OD value recovery were performed to evaluate the degree of cell damage caused by quenching at single cell level and at integral level respectively.The tested results indicated that the degree of damage to cells caused by cold sodium chloride solution was less than 5%.The peak quantity and the total ion intensity detected by LC-TOF in low collision energy were used to evaluate extraction effects.Three different cell membrane penetrability modes and 4 kinds of extraction solvents were investigated and compared.The results showed that the technique of liquid nitrogen freezing combined with ultrasonic processing for cell membrane penetrability and a cold aqueous solution of methanol(MeOH/H 2O,1∶1,V/V,4℃)for extraction of metabolites had the best extraction effect(peak quantity was greater than or equal to 10^5,and total ion intensity was in the range of 10^6-10^7).Therefore,in this work,the freeze drying,grinding with liquid nitrogen and ultrasonic extraction were combined to extract metabolites.In this way,it effectively promoted cell lysis and improved the efficiency of extraction.The result of synthetic analysis showed that the method proposed here could meet the requirements of the metabonomics analysis of Escherichia coli.