CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Methods. 2020 May 21;12(19):2491-2498. doi: 10.1039/c9ay02753j.
With the wide application of cell metabolomics in many research areas, there is a need to develop an effective procedure for adherent mammalian cell metabolomics that allows for accurate determination of intracellular metabolite levels and easy comparison between multiple studies of a similar application. Here we aimed to compare the efficiencies of different cell harvesting methods and metabolite extraction methods in sample preparation procedures, and to provide a cell sample processing protocol which focuses on maximizing metabolite recovery ranging from polar to lipidic ones. A systematical evaluation of 4 cell harvesting methods and 4 extraction methods was conducted based on human hepatoma HepG2 cells. The impact of different methods on the recoveries of 11 different categories of metabolites was further investigated. The water disruption sample harvesting method provided superior performance to the other 3 harvesting methods, trypsinization, scraping in phosphate buffered saline, and direct solvent scraping, with respect to the recoveries of polar metabolites and lipids. Among the 4 extraction methods, the novel two-phase solvent system extraction method with both methyl tert-butyl ether (MTBE) and 75% 9 : 1 methanol : chloroform showed an absolute advantage with high extraction efficiency for global metabolomics. We showed a metabolite-specific impact of the harvesting method and extraction method on metabolite concentrations. The water disruption sample collection combined with novel two-phase solvent system extraction enabled simultaneous profiling of lipids and metabolites with mixed polarity for sample preparation. Our approach may open up new perspectives toward large-scale comprehensive metabolomic analyses of adherent mammalian cell samples.
随着细胞代谢组学在许多研究领域的广泛应用，需要开发一种有效的贴壁哺乳动物细胞代谢组学处理程序，以准确测定细胞内代谢物水平，并便于对类似应用的多项研究进行比较。在这里，我们旨在比较不同细胞收获方法和代谢物提取方法在样品制备程序中的效率，并提供一种专注于最大限度回收从极性到脂质的代谢物的细胞样品处理方案。我们基于人肝癌 HepG2 细胞对 4 种细胞收获方法和 4 种提取方法进行了系统评价。进一步研究了不同方法对 11 种不同类别代谢物回收率的影响。与胰酶消化、磷酸盐缓冲液刮取和直接溶剂刮取这 3 种收获方法相比，水裂解法在极性代谢物和脂质的回收率方面表现出了更好的性能。在 4 种提取方法中，新型两相溶剂系统提取方法（同时使用甲基叔丁基醚 (MTBE) 和 75% 9:1 甲醇：氯仿）具有绝对优势，对全局代谢组学具有很高的提取效率。我们展示了收获方法和提取方法对代谢物浓度的具体影响。水裂解法与新型两相溶剂系统提取相结合，可用于同时对混合极性的细胞样本进行脂质和代谢物的分析。我们的方法可能为贴壁哺乳动物细胞样品的大规模综合代谢组学分析开辟新的视角。
