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dc.contributor.authorYuan, Min
dc.contributor.authorKremer, Daniel M.
dc.contributor.authorHuang, He
dc.contributor.authorBreitkopf, Susanne B.
dc.contributor.authorBen-Sahra, Issam
dc.contributor.authorManning, Brendan
dc.contributor.authorLyssiotis, Costas A.
dc.contributor.authorAsara, John
dc.date.accessioned2022-06-06T16:24:44Z
dc.date.issued2019-02
dc.identifier.citationYuan, Min, Daniel M. Kremer, He Huang, Susanne B. Breitkopf, Issam Ben-Sahra, Brendan Manning, Costas A. Lyssiotis et al. "Ex Vivo and in Vivo Stable Isotope Labelling of Central Carbon Metabolism and Related Pathways With Analysis by LC–MS/MS." Nature Protocols 14, no. 2 (2019): 313-330. DOI: 10.1038/s41596-018-0102-x
dc.identifier.issn1754-2189en_US
dc.identifier.issn1750-2799en_US
dc.identifier.urihttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37371890*
dc.description.abstractTargeted tandem mass spectrometry (LC–MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (13C) and nitrogen-15 (15N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. This protocol represents an extension of a previously published protocol for targeted metabolomics of unlabeled species and has been used extensively in tracing the metabolism of nutrients such as 13C-labeled glucose, 13C-glutamine and 15N-glutamine in a variety of biological settings (e.g., cell culture experiments and in vivo mouse labelling via i.p. injection). SRM signals are integrated to produce an array of peak areas for each labelling form that serve as the output for further analysis. The processed data are then used to obtain the degree and distribution of labelling of the targeted molecules (termed fluxomics). Each method can be customized on the basis of known unlabeled Q1/Q3 SRM transitions and adjusted to account for the corresponding 13C or 15N incorporation. The entire procedure takes ~6–7 h for a single sample from experimental labelling and metabolite extraction to peak integration.en_US
dc.language.isoen_USen_US
dc.publisherSpringer Science and Business Media LLCen_US
dc.relationNature Protocolen_US
dc.relation.isversionofdoi:10.1038/s41596-018-0102-xen_US
dc.relation.hasversionhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382369/en_US
dash.licenseMETA_ONLY
dc.subjectResearch Subject Categories::NATURAL SCIENCES::Chemistry::Biochemistryen_US
dc.subjectResearch Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biologyen_US
dc.titleEx Vivo and in Vivo Stable Isotope Labelling of Central Carbon Metabolism and Related Pathways With Analysis by LC–MS/MSen_US
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden_US
dc.relation.journalNature Protocolsen_US
dash.depositing.authorAsara, John
dash.waiver2018-10-03
dc.date.available2022-06-06T16:24:44Z
dash.affiliation.otherHarvard T.H. Chan School of Public Healthen_US
dc.identifier.doi10.1038/s41596-018-0102-x
dc.source.journalNat Protoc
dash.waiver.reasonThis was a collaboration with an investigator at BIDMC, who is corresponding author and chose the journal, which is also the most appropriate journal for this particular study.en_US
dash.source.volume14en_US
dash.source.page313-330en_US
dash.source.issue2en_US
dash.contributor.affiliatedAsara, John
dash.contributor.affiliatedManning, Brendan


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