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An automated GCxGC-TOF-MS protocol for batch-wise extraction and alignment of mass isotopomer matrixes from differential C-13-labelling experiments : a case study for photoautotrophic-mixotrophic grown Chlamydomonas reinhardtii cells

  • Two dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC-TOF-MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC-TOF-MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of 13 C-labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzedTwo dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC-TOF-MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC-TOF-MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of 13 C-labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzed without separation of the medium. The growth medium was adapted to this rapid sampling protocol to avoid interference with GCxGC-TOF-MS analysis. To analyse batches of samples a new software tool, MetMax, was implemented which extracts the isotopomer matrix from stable isotope labelling experiments together with the first and second retention index (RI1 and RI2). To exploit RI1 and RI2 for metabolite identification we used the Golm metabolome database (GMD [1] with RI1/ RI2-reference spectra and new search algorithms. Using those techniques we analysed the dynamics of (CO2)-C-13 and C-13- acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotrophic and mixotrophic growth conditions.‚Ķshow moreshow less

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Metadaten
Author:Stefan Kempa, Jan Hummel, Thorsten Schwemmer, Matthias Pietzke, Nadine Strehmel, Stefanie Wienkoop, Joachim Kopka, Wolfram Weckwerth
URL:http://www3.interscience.wiley.com/cgi-bin/jhome/5007687
DOI:https://doi.org/10.1002/jobm.200800337
ISSN:0233-111X
Document Type:Article
Language:English
Year of first Publication:2009
Year of Completion:2009
Release Date:2017/03/25
Source:Journal of basic microbiology. - ISSN 0233-111X. - 49 (2009), 1, S. 82 - 91
Organizational units:Extern / Extern
Peer Review:Referiert