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

Author(s)

Stefan Kempa, Jan Hummel, Thorsten Schwemmer, Matthias Pietzke, Nadine Strehmel, Stefanie Wienkoop, Joachim Kopka, Wolfram Weckwerth

Abstract

Two dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGCTOF-

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

13C-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 13CO2 and 13Cacetate

uptake in Chlamydomonas reinhardtii cells in two different steady states namely

photoautotroph and mixotroph growth conditions.

Organisation(s)

External organisation(s)

Max-Planck-Institut für Molekulare Pflanzenphysiologie

Journal

Journal of Basic Microbiology: an international journal on biochemistry, physiology, genetics, morphology and ecology of microorganisms

Volume

Pages

82-91

No. of pages

ISSN

0233-111X

Publication date

2009

Peer reviewed

Yes

Austrian Fields of Science 2012

1030 Physics, Astronomy, 106022 Microbiology, 106031 Plant physiology

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