Mass Spectrometry-Based Untargeted Metabolomics and Lipidomics Platforms to Analyze Cell Culture Extracts

Abstract

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.

Keywords: Cell culture extracts; HepaRG cells; High-resolution mass spectrometry; Hydrophilic interaction liquid chromatography; Quality assurance and quality control; Reversed-phase liquid chromatography; Untargeted lipidomics; Untargeted metabolomics.