Development of a simple and efficient method of harvesting and lysing adherent mammalian cells for chemical isotope labeling LC-MS-based cellular metabolomics

Abstract

Chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) has a potential to become a very powerful platform for comprehensive and quantitative analysis of metabolites in cellular metabolomics. While CIL LC-MS with rationally designed reagents for labeling can provide much enhanced detection and quantification of metabolites, pre-analytical process such as cell harvest and cell lysis is also a critical step in the overall workflow for metabolomic analysis. In this study, we examined various combinations of cell harvest and cell lysis methods in order to develop a simple and efficient method for harvesting and lysing adherent mammalian cells tailored to CIL LC-MS-based cellular metabolomics. We evaluated and compared two cell harvest methods (physical scraping and trypsinization) and two cell lysis methods (glass-bead-assisted lysis and freeze-thaw-cycle lysis). We used two types of commonly used mammalian cells, HeLa and MCF-7 cells, to cross-validate the findings. LC-UV quantification of the labeled metabolites after dansylation of cell extracts was used to compare the overall cell harvest and cell lysis efficiencies among different combination methods. It was found that scraping combined with freeze-thaw-cycle lysis gave the highest total metabolite concentration. We also performed multivariate and univariate analyses of the amine/phenol submetabolome data generated from different preparations to investigate the impact of cell harvest and lysis methods on cellular metabolome profiles. Comparing to scraping, trypsinization caused more significant metabolome changes likely due to metabolite leakage and metabolite level changes. The cellular metabolomes obtained from the two lysis methods were found to be similar; however, freeze-thaw-cycle lysis gave a higher lysis efficacy, compared to the glass bead method. We concluded that the combination of scraping and freeze-thaw-cycle was optimal for harvesting and lysing adherent mammalian cells for CIL LC-MS metabolomics.