基于液质联用的人血清代谢组学样品前处理方法研究

A study of human serum preparation methods for metabolomic analysis based on liquid chromatography-mass spectrometry

目的建立一种稳定、可靠且适合大样本分析的血清代谢组学前处理方法。方法采用超高效液相色谱-四级杆飞行时间串联质谱(ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry,UPLCQTOF-MS)分析方法,对经三倍乙腈沉淀处理(方法一)后直接进样、三倍乙腈沉淀后再通过浓缩复溶方式处理(方法二)后进样及一倍乙腈沉淀后再通过超滤管截留大分子方式处理(方法三)后进样的血清样品前处理方法进行对比分析,同时利用质控样品对仪器精密度、稳定性、重复性进行考察。结果 ESI+和ESI-扫描模式下,方法二可检测到的代谢物特征数最多,方法三次之,而方法一最少。方法一与方法三在ESI+和ESI-扫描模式下测得离子峰的峰强度均值比较差异均无统计学意义(均P>0.05),但均大于方法二(均P<0.05);且方法二和方法三大部分离子峰的峰强度变异系数(coefficient of variation,CV)值<25.00%,而方法一中大部分离子峰的峰强度CV值>25.00%,说明方法三的代谢物提取重复性更好,既能保证检测到的特征总数,又能保证检测到的代谢物的峰强度。实验过程中仪器的精密度、稳定性、重复性均较好。结论一倍乙腈沉淀蛋白后再通过超滤管截留大分子的血清前处理方法操作简便、重复性较好,且能更全面保留和检测血清样品中的小分子化合物,适用于血清代谢组学大批量样本分析。

Objective To establish a stable,reliable and suitable preparation method of serum metabolomics for the analysis of large samples.Methods The ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)method was utilized to comparatively analyze the preparation methods of human serum for metabolomic analysis,including direct three-fold acetonitrile precipitation(Method 1),concentration and reconstitution after three-fold acetonitrile precipitation(Method2),and one-fold acetonitrile precipitation(Method 3),and then the biological quality control sample was used to investigate the precision,stability and repeatability of instruments.Results In ESI+and ESI-scanning,Method 2 yielded the largest characteristic number of metabolites,Method 1 yielded the least number,and Method 3 was in between.No statistically significant difference in the mean peak intensity of the ion peaks was found between Method 1 and Method 3 in the ESI+and ESI-scanning(all P>0.05),but the results of both methods were greater than that of Method 2(all P<0.05).The peak intensity coefficient of variation(CV)values of the most ion peaks of Method 2 and Method 3 were less than 25.00%,whereas that of Method 1 was more than 25.00%.The results indicated that the metabolite extraction of Method 3 had better repeatability,and this method could guarantee the proper detection of both the total characteristic number and the peak intensity of metabolites.The precision,stability and repeatability of the instrument during the experiment were all good.Conclusion The serum preparation method that protein molecules are first one-fold acetonitrile precipitated and then intercepted through the ultrafiltration tube can comprehensively retain and detect small molecule compounds in serum samples with a simple and nice reproducible way,and is suitable for metabolomics research.