血浆代谢组同时定性定量分析方法研究

数据非依赖的质谱采集是近年来发展的一种新型多级质谱分析方法,只需一次进样便可同时快速获取所有母离子及其子离子信息。本研究以肺癌血浆样本为研究对象,采用数据非依赖的SWATH(sequential windowed acquisition of all theoretical fragment ions)采集技术,建立了液相色谱-串联质谱法(LC-MS/MS)同时定性定量分析血浆代谢组。基于代谢物数据库识别比对,成功识别了93个代谢物,实现了代谢物的定性分析。对成功识别的代谢物建立定量分析方法并进行方法学考察。结果表明,其中90个代谢物的线性相关系数大于0.99,定量限在1.25~12000μg/L之间;其中有86个代谢物可以满足生物样本定量分析的要求,其连续4天的日内精密度和日间精密度均小于20%;在4℃下,96 h内的放置稳定性在0.62%~19.35%之间。该方法的覆盖率高,能同时快速准确地对肺癌血浆中癌症相关代谢物进行定性定量分析,也适用于其他生物样品,可以为定量代谢组学分析提供重要的方法学平台。

Development of Simultaneous Qualitation and Quantification of Plasma Metabolome Based on Sequential Windowed Acquisition of All Theoretical Fragment Ions (SWATH)

Data independent acquisition (DIA) mode, is a novel multi-stage mass spectrometry (MS) acquisition technology and it can achieve rapid detection of full MS and all product ion spectra in a single liquid chromatography coupled with mass spectrometry (LC/MS) run. Sequential Windowed Acquisition of all Theoretical fragment ions (SWATH) is one of DIA mode. The scanning range of SWATH acquisition is divided into multiple continuous intervals which contains a certain width (usually 25 u), and all ions in the interval are fragmented and quickly scanned in order to acquire all MS and MS/MS information. On one hand, it can provide relatively simple MS/MS spectra of ions due to its relatively narrow m/z isolation window when comparing with other DIA mode, so it is widely used in untargeted metabonomics analysis. On the other hand, it can exhibit comparative sensitivity and quantitative accuracy when comparing with multiple reaction monitoring (MRM) and can be used for quantitative analysis. Thus, SWATH acquisition can show the advantage of high sensitivity, high coverage and high scanning speed. In this study, a simultaneous qualitative and quantitative analysis of multiple metabolites in the plasma was developed based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with SWATH acquisition. 93 metabolites in the plasma can be successfully identified by home-made metabolites database searching which consists of metabolites related to cancers based on our previous metabonomics studies, thus achieving untargeted analysis. Quantification method was further developed for identified metabolites and methodology was examined. The analysis result shows that calibration curves were linear (the linearity of 95% metabolites more than 0.99) with limits of detection from 1.25 to 12 000 μg/L. In order to ensure the reliability of method, accuracy and stability of the method were further investigated, and 86 of metabolites in the plasma can achieve the quantification requirements in biological samples; Intra-day relative standard deviation (RSD) and inter-day RSD are both lower than 20%, and stability for 96 hours at 4 ℃ is from 0.62% to 19.35%. It’s worth noting that for metabolites with low content in the plasma or serious matrix interference, quantitation by its product ion can exhibit better linearity, wider dynamic ranges and lower limit of quantitation. The established method provides a reliable and rapid method for identifying and quantifying multiple metabolites simultaneously in the plasma with high coverage and high sensitivity, and it is also applicative in the analysis of other biological samples.