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dc.contributor.authorThorfinnsdottir, Lilja Brekke
dc.contributor.authorGarcia Calvo, Laura
dc.contributor.authorBø, Gaute Hovde
dc.contributor.authorBruheim, Per
dc.contributor.authorRøst, Lisa Marie Ivarsdatter
dc.date.accessioned2023-09-28T14:22:40Z
dc.date.available2023-09-28T14:22:40Z
dc.date.created2023-02-01T09:04:23Z
dc.date.issued2023
dc.identifier.issn2218-1989
dc.identifier.urihttps://hdl.handle.net/11250/3092781
dc.description.abstractPrecise and accurate quantification is a prerequisite for interpretation of targeted metabolomics data, but this task is challenged by the inherent instability of the analytes. The sampling, quenching, extraction, and sample purification conditions required to recover and stabilize metabolites in representative extracts have also been proven highly dependent on species-specific properties. For Escherichia coli, unspecific leakage has been demonstrated for conventional microbial metabolomics sampling protocols. We herein present a fast filtration-based sampling protocol for this widely applied model organism, focusing on pitfalls such as inefficient filtration, selective loss of biomass, matrix contamination, and membrane permeabilization and leakage. We evaluate the effect of and need for removal of extracellular components and demonstrate how residual salts can challenge analytical accuracy of hyphenated mass spectrometric analyses, even when sophisticated correction strategies are applied. Laborious extraction procedures are bypassed by direct extraction in cold acetonitrile:water:methanol (3:5:2, v/v%), ensuring compatibility with sample concentration and thus, any downstream analysis. By applying this protocol, we achieve and demonstrate high precision and low metabolite turnover, and, followingly, minimal perturbation of the inherent metabolic state. This allows us to herein report absolute intracellular concentrations in E. coli and explore its central carbon metabolome at several commonly applied cultivation conditions.en_US
dc.language.isoengen_US
dc.publisherMDPIen_US
dc.rightsNavngivelse 4.0 Internasjonal*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/deed.no*
dc.titleOptimized Fast Filtration-based Sampling and Extraction Enables Precise and Absolute Quantification of the Escherichia coli Central Carbon Metabolomeen_US
dc.title.alternativeOptimized Fast Filtration-based Sampling and Extraction Enables Precise and Absolute Quantification of the Escherichia coli Central Carbon Metabolomeen_US
dc.typePeer revieweden_US
dc.typeJournal articleen_US
dc.description.versionpublishedVersionen_US
dc.source.volume13en_US
dc.source.journalMetabolitesen_US
dc.source.issue2en_US
dc.identifier.doi10.3390/metabo13020150
dc.identifier.cristin2121519
dc.relation.projectTrond Mohn stiftelse: TMS2019TMT05en_US
dc.relation.projectNorges teknisk-naturvitenskapelige universitet: Muliggjørende teknologi Bioteknologien_US
cristin.ispublishedtrue
cristin.fulltextoriginal
cristin.qualitycode1


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