Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry

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dc.contributor.author Smart, KF en
dc.contributor.author Aggio, Raphael en
dc.contributor.author Van Houtte, JR en
dc.contributor.author Villas-Boas, Silas en
dc.date.accessioned 2012-04-01T21:37:49Z en
dc.date.issued 2010-01-01 en
dc.identifier.citation NATURE PROTOCOLS 5(10):1709-1729 01 Jan 2010 en
dc.identifier.issn 1754-2189 en
dc.identifier.uri http://hdl.handle.net/2292/16270 en
dc.description.abstract This protocol describes an analytical platform for the analysis of intra-and extracellular metabolites of microbial cells (yeast, filamentous fungi and bacteria) using gas chromatography-mass spectrometry (GC-MS). The protocol is subdivided into sampling, sample preparation, chemical derivatization of metabolites, GC-MS analysis and data processing and analysis. This protocol uses two robust quenching methods for microbial cultures, the first of which, cold glycerol-saline quenching, causes reduced leakage of intracellular metabolites, thus allowing a more reliable separation of intra-and extracellular metabolites with simultaneous stopping of cell metabolism. The second, fast filtration, is specifically designed for quenching filamentous micro-organisms. These sampling techniques are combined with an easy sample-preparation procedure and a fast chemical derivatization reaction using methyl chloroformate. This reaction takes place at room temperature, in aqueous medium, and is less prone to matrix effect compared with other derivatizations. This protocol takes an average of 10 d to complete and enables the simultaneous analysis of hundreds of metabolites from the central carbon metabolism (amino and nonamino organic acids, phosphorylated organic acids and fatty acid intermediates) using an in-house MS library and a data analysis pipeline consisting of two free software programs (Automated Mass Deconvolution and Identification System (AMDIS) and R). en
dc.language English en
dc.publisher NATURE PUBLISHING GROUP en
dc.relation.ispartofseries Nature Protocols en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. Details obtained from: http://www.sherpa.ac.uk/romeo/issn/1754-2189/ en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.subject Science & Technology en
dc.subject Life Sciences & Biomedicine en
dc.subject Biochemical Research Methods en
dc.subject Biochemistry & Molecular Biology en
dc.subject INTRACELLULAR METABOLITES en
dc.subject FUNCTIONAL GENOMICS en
dc.subject EXTRACELLULAR METABOLOMICS en
dc.subject QUANTITATIVE METABOLOMICS en
dc.subject SACCHAROMYCES-CEREVISIAE en
dc.subject SYSTEMS BIOLOGY en
dc.subject GC-MS en
dc.subject IDENTIFICATION en
dc.subject YEAST en
dc.subject QUANTIFICATION en
dc.title Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry en
dc.type Journal Article en
dc.identifier.doi 10.1038/nprot.2010.108 en
pubs.issue 10 en
pubs.begin-page 1709 en
pubs.volume 5 en
dc.rights.holder Copyright: NATURE PUBLISHING GROUP en
dc.identifier.pmid 20885382 en
pubs.author-url http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=000282369100011&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=6e41486220adb198d0efde5a3b153e7d en
pubs.end-page 1729 en
dc.rights.accessrights http://purl.org/eprint/accessRights/RestrictedAccess en
pubs.subtype Article en
pubs.elements-id 165725 en
pubs.record-created-at-source-date 2012-04-02 en
pubs.dimensions-id 20885382 en


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