Toxicological Centre, University of Antwerp, Antwerp, Belgium.
Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium.
Methods Mol Biol. 2023;2571:189-206. doi: 10.1007/978-1-0716-2699-3_19.
Metabolites represent the most downstream level of the cellular organization. Hence, an in vitro untargeted metabolomics approach is extremely valuable to deepen the understanding of how endogenous metabolites in cells are altered under a given biological condition. This chapter describes a robust liquid chromatography-high-resolution mass spectrometry-based metabolomics and lipidomics platform applied to cell culture extracts. The analytical workflow includes an optimized sample preparation procedure to cover a wide range of metabolites using liquid-liquid extraction and validated instrumental operation procedures with the implementation of comprehensive quality assurance and quality control measures to ensure high reproducibility. The lipidomics platform is based on reversed-phase liquid chromatography for the separation of slightly polar to apolar metabolites and covers a broad range of lipid classes, while the metabolomics platform makes use of two hydrophilic interaction liquid chromatography methods for the separation of polar metabolites, such as organic acids, amino acids, and sugars. The chapter focuses on the analysis of cultured HepaRG cells that are derived from a human hepatocellular carcinoma; however, the sample preparation and analytical platforms can easily be adapted for other types of cells.
代谢物代表细胞组织的最下游水平。因此，体外无靶向代谢组学方法对于深入了解在给定的生物条件下细胞内的内源性代谢物如何发生变化非常有价值。本章描述了一个强大的基于液相色谱-高分辨率质谱的代谢组学和脂质组学平台，应用于细胞培养提取物。分析工作流程包括一个优化的样品制备程序，使用液液萃取覆盖广泛的代谢物范围，并验证了仪器操作程序，实施了全面的质量保证和质量控制措施，以确保高重现性。脂质组学平台基于反相液相色谱法分离稍极性到非极性代谢物，涵盖广泛的脂质类别，而代谢组学平台则利用两种亲水相互作用液相色谱方法分离极性代谢物，如有机酸、氨基酸和糖。本章重点介绍了源自人肝癌的 HepaRG 细胞的分析；然而，样品制备和分析平台可以很容易地适应其他类型的细胞。
