亲水作用色谱/质谱联用方法用于大肠杆菌代谢组分析

Hydrophilic interaction liquid chromatography coupled with mass spectrometry for metabolomic analysis of Escherichia coli

建立了两性离子亲水作用色谱/质谱联用方法用于大肠杆菌胞内极性代谢物的分离分析。选取52个代表性极性物质对方法进行考察，发现此方法有较好的线性范围，且大部分物质最低检测限均在 ng/mL 数量级。平行制备6份样品进行分析，结果显示85％以上代谢物峰面积的 RSD值小于30％。6个内标物质在低、中、高3个浓度下的日内精密度（ RSD）均小于20％，大部分物质的相对回收率都在可接受的范围内（70％~130％）。把此方法用于yfcC基因改造的3株大肠杆菌代谢组分析，发现一些小肽、氨基酸、核苷、有机酸、磷脂等物质在基因改造后发生明显变化。此研究结果表明，建立的两性离子亲水作用色谱/质谱方法检测到的物质化学性质分布广，跨越了极性磷脂到小肽的各个范围，且具有良好的重复性、稳定性和适用性。

The zwitterionic hydrophilic interaction liquid chromatography coupled with mass spectrometry（ ZIC-HILIC/MS）method was developed and applied for studying the metabolome of Escherichia coli. In this method,the linear ranges of the 52 representative compounds were between 2-6 orders of magnitude. Moreover the lowest limits of detection of most compounds were at ng/mL levels. The analysis for six samples prepared in parallel showed that the method had good reproducibility with more than 85% metabolites with the RSDs of peak area less than 30%. The precision of the method was tested by evaluating the relative recoveries of six internal standards at the low,middle and high concentrations. The results exhibited that the intraday precisions（ RSDs）of the six compounds in the same concentrations were less than 20%. The recoveries of most compounds were accepted in the range of 70%-130%. The results of metabo-lomic analysis of three Escherichia coli strains with genetic modified yfcC gene showed that some small peptides, amino acids, nucleosides, organic acids and phospholipids changed differentially after the yfcC gene modification. These results indicated that the ZIC-HILIC/MS method could detect diverse metabolites and be used for metabolomic analysis purpose with good reproducibility and stability.