Sample Preparation and Reporting Standards for Metabolomics of Adherent Mammalian Cells
Metabolomics is an analytical technique that investigates the small molecules present within a biological system. Metabolomics of cultured cells allows profiling of the metabolic chemicals involved in a cell type-specific system and the response of that m
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uction Metabolomics studies frequently state that the metabolome is a closer reflection of the phenotype of an organism, tissue, or cell than other “omics analyses,” such as proteomics, transcriptomics, and genomics [1–3]. The small molecules that are shuffled around the vast network of metabolic pathways in a biological system are referred to as metabolites, with the “metabolome” made up of the many thousands of different metabolites and their relative levels of abundance. The goal of metabolomic profiling is to measure changes in the metabolome of a given system in response to a challenge to normal cellular homeostasis, whether physical, chemical, environmental, or other external stressor.
Angelo D’Alessandro (ed.), High-Throughput Metabolomics: Methods and Protocols, Methods in Molecular Biology, vol. 1978, https://doi.org/10.1007/978-1-4939-9236-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Sarah Hayton et al.
Metabolomics has recently become an attractive application for untargeted, high-throughput screening analyses [4]. However, due to the data-rich, hypothesis-generating outcomes of untargeted metabolomics, it is understandable that such an approach might not be attractive to some investigators, particularly if a limited number of clinical or animal samples are available. In particular, handling the vast amounts of data can complicate meaningful biological interpretation of the data. This can be partly addressed by the use of cultured mammalian cells, as opposed to animal- based samples, to more easily accommodate re-visiting of the sample set if novel or previously unknown metabolites are highlighted by an untargeted study. The number of controls and replicates can be easily manipulated in design of cell culture-based experiments, benefiting the development, validation, and standardization of untargeted, high-throughput metabolomic studies. The importance of experimental design and standardized reporting requirements for these studies of cultured mammalian cells has been previously reviewed across a large number of published protocols [5, 6]. The focus of this protocol is to give the investigator a sound, high-throughput procedure to follow for handling and preparing cultured cell samples preceding instrumental analysis for metabolomics, which will have minimal possible interference on the detectable metabolome. It is designed so that any of the multiple platforms currently used for metabolomics can be applied to the prepared samples thereafter (e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry). Here we provide a standardized protocol for untargeted metabolomic analysis applied to any cultured mammalian cell line, highlighting the importance of adequate reporting of culture conditions to allow for meaningful biological interpretation of metabolomic data and comparison of results across multiple studies.
2 Materials 2.1 Cell Lines
Cell lines should be sourced from reputable sources such as the European Collection of Cell Cultures (E
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