An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines
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ORIGINAL ARTICLE
An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines Marina Wright Muelas1 · Ivayla Roberts1 · Farah Mughal2,3 · Steve O’Hagan3,4 · Philip J. Day1,5 · Douglas B. Kell1 Received: 4 June 2020 / Accepted: 18 September 2020 © The Author(s) 2020
Abstract Introduction It is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites. Objectives Blood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to detect and identify metabolites present in serum, but to also establish a method capable of measure their uptake and secretion by different cell lines. Methods We develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the ‘exometabolome’ or metabolic footprint). Results Our method measures some 4000–5000 metabolic features in both positive and negative electrospray ionisation modes. We show that the metabolic footprints of different cell lines differ greatly from each other. Conclusion Our new, 15-min untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum. This will enable future research to study these differences in multiple cell lines that will relate this to transporter expression, thereby advancing our knowledge of transporter substrates, both natural and xenobiotic compounds. Keywords Human serum · Untargeted metabolomics · Transporters · LC-MS/MS · Orbitrap · Cell culture
1 Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11306-020-01725-8) contains supplementary material, which is available to authorized users.
One of the great unsolved problems of modern biology concerns the substrates of membrane transporters (CesarRazquin et al. 2018; Cesar-Razquin et al. 2015; Girardi et al. 2020; Superti-Furga et al. 2020), many of which remain ‘orphans’ (i.e. with unknown substrates), often despite the
* Marina Wright Muelas m.wright‑[email protected]
3
The Manchester Institute of Biotechnology, 131, Princess St, Manchester M1 7DN, UK
* Douglas B. Kell [email protected]
4
Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PL, UK
5
Novo Nordisk Foundation Centre for Biosustainability, Technical Univers
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