Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK.
School of Chemistry, The Manchester Institute of Biotechnology, 131, Princess St, Manchester, M1 7DN, UK.
Metabolomics. 2020 Oct 7;16(10):107. doi: 10.1007/s11306-020-01725-8.
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.
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.
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).
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.
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.
人们普遍但错误地认为，药物通过穿过质膜和其他膜的磷脂双层部分进入细胞。然而，大量证据表明，事实并非如此，药物通过搭乘其他天然分子的转运体跨越生物膜，而这些药物在结构上与这些天然分子相似。非靶向代谢组学可以提供一种确定这些代谢物差异摄取的方法。
血清中含有数千种分子，是生物相关代谢物的方便来源。我们的目的是检测和识别血清中存在的代谢物，但也要建立一种能够测量不同细胞系摄取和分泌这些代谢物的方法。
我们开发了一种非靶向 LC-MS/MS 方法来检测人血清中存在的广泛化合物。我们将其应用于分析几种人细胞系中血清中代谢物摄取和分泌的时间过程，通过分析代表细胞外相（“外代谢组”或代谢足迹）的血清中的变化来实现。
我们的方法在正离子和负离子电喷雾离子化模式下测量了大约 4000-5000 种代谢特征。我们表明，不同细胞系的代谢足迹彼此之间存在很大差异。
我们新的 15 分钟非靶向代谢组学方法允许在孵育血清后对细胞系摄取血清化合物的差异进行稳健和方便的测量。这将使未来的研究能够在多种细胞系中研究这些差异，并将其与转运体表达相关联，从而推进我们对转运体底物的了解，包括天然和外源性化合物。
