Optimization of the sample preparation method for adherent cell metabolomics based on ultra-performance liquid chromatography coupled to mass spectrometry

Sample preparation plays a crucial role in generating data with satisfactory quality and stability for metabolomics studies, especially those conducted on cells cultured in vitro. Thus, to carry out a preparation procedure with a low risk of systematic error, the operating steps for a cell metabolomics study were optimized step by step, which include cell quenching, harvesting, cracking, leveling and extraction. First, the metabolites presenting high amounts, including amino acids, acylcarnitines and phosphatidylcholines, were screened out from the other metabolites. Consequently, six acylcarnitines revealed much better concentration-response linearity (R2＞0.95) than others. Thus, the candidate methods for each step were evaluated via the comparison of the relative standard deviations of the screened metabolites among cell duplicates. Liquid nitrogen for quenching, trypsinization for harvesting, sonication for cracking, cell counting for leveling, and the sole use of methanol for metabolite extraction were the methods presenting the best repeatability compared with the corresponding counterpart methods. Additionally, samples cracked with sonication as well as those quenched with liquid nitrogen revealed significant higher intensities than corresponding counterpart methods. To further explore the metabolic disturbances caused by trypsinization, the metabolic profile of trypsinization-treated cells was compared with those harvested by scraping using multivariate analysis and metabolic enrichment analysis. Glycine, serine and threonine metabolism as well as glycerophospholipid metabolism showed the most significant variations due to the biochemically associated metabolites mapped in them. In conclusion, this study would prospectively provide analysts and biologists with a helpful reference in choosing methods for sample preparation in cell metabolomics studies.