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An automated GCxGC‐TOF‐MS protocol for batch‐wise extraction and alignment of mass isotopomer matrixes from differential 13 C‐labelling experiments: a case study for photoautotrophic‐mixotrophic grown Chlamydomonas reinhardtii cells
Author(s) -
Kempa Stefan,
Hummel Jan,
Schwemmer Thorsten,
Pietzke Matthias,
Strehmel Nadine,
Wienkoop Stefanie,
Kopka Joachim,
Weckwerth Wolfram
Publication year - 2009
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.200800337
Subject(s) - chlamydomonas reinhardtii , time of flight mass spectrometry , metabolome , chromatography , mass spectrometry , metabolomics , isotopomers , chemistry , biochemistry , mutant , ion , organic chemistry , gene , ionization , molecule
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 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 13 CO 2 and 13 C‐acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotroph and mixotroph growth conditions. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)