Abstract
IntroductionInterest in cell culture metabolomics has increased greatly in recent years because of its many potential applications and advantages (e.g., in toxicology). The first critical step for exploring the cellular metabolome is sample preparation. For metabolomics studies, an ideal sample preparation would extract a maximum number of metabolites and would enable reproducible, accurate analysis of a large number of samples and replicates. In addition, it would provide consistent results across several studies over a relatively long time frame.ObjectivesThis study was conducted to evaluate the impact of sample preparation strategies on monitoring intracellular metabolite responses, highlighting the potential critical step(s) in order to finally improve the quality of metabolomics studies.MethodsThe sample preparation strategies were evaluated by calculating the sample preparation effect, matrix factor, and process efficiency (PE) for 16 tobacco exposition-related metabolites, including nicotine, nicotine-derived nitrosamine ketone, their major metabolites, and glutathione, using isotopically-labelled internal standards. Samples were analyzed by liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS).ResultsA sample drying step increased losses or variability for some selected metabolites. By avoiding evaporation, good sample preparation recovery was obtained for these compounds. For some metabolites, the cell or culture type impacted PE and matrix factor.ConclusionIn our sample preparation protocol, the drying–reconstitution step was identified as the main cause of metabolite losses or increased data variability during metabolomics analysis by LC-HRMS. Furthermore, PE was affected by the type of matrix. Isotopologue internal standards fully compensate losses or enhancements.
Highlights
Interest in cell culture metabolomics has increased greatly in recent years because of its many potential applications and advantages
Sample preparation protocols can be influenced by cell culture properties as well as cell types, the physico-chemical properties of the metabolites, and the techniques used for their detection. 3D cell cultures were used for this investigation because they are routinely used for toxicological assessment, and a new extraction approach was required compared with 2D cell cultures
To evaluate the precision of a non-discriminative analytical approach for intracellular metabolite analysis, sample preparation effect, matrix factor, and process efficiency (PE) were calculated for 16 metabolites of interest and eight internal standards (ISTD) related to our research scope
Summary
Interest in cell culture metabolomics has increased greatly in recent years because of its many potential applications and advantages (e.g., in toxicology). An ideal sample preparation would extract a maximum number of metabolites and would enable reproducible, accurate analysis of a large number of samples and replicates It would provide consistent results across several studies over a relatively long time frame. Characterization of the cellular metabolome, represented by intermediates and end products of all enzymatic reactions in a cell, enables the evaluation of cellular biochemical activity/networks This information can predict molecular physiology and phenotypic changes in an organism (Töpfer et al 2015). (ii) A targeted approach is focused on a limited list of metabolites In this case, sample preparation and analytical methods are optimized for specific compounds, which are absolutely quantified using calibration curves and internal standards for each metabolite. Sample preparation and analytical methods are optimized for specific compounds, which are absolutely quantified using calibration curves and internal standards for each metabolite. (iii) A semi-targeted approach
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