Assessment of sampling strategies for gas chromatography-mass spectrometry (GC-MS) based metabolomics of cyanobacteria.
Metabolomics is the comprehensive analysis of the small molecules that compose an organism's metabolism. The main limiting step in microbial metabolomics is the requirement for fast and efficient separation of microbes from the culture medium under conditions in which metabolism is rapidly halted. In this article we compare three different sampling strategies, quenching, filtering, and centrifugation, for arresting the metabolic activities of two morphologically diverse cyanobacteria, the unicellular Synechocystis sp. PCC 6803 and the filamentous Nostoc sp. PCC 7120 for GC-MS analysis. We demonstrate that each sampling technique produces internally consistent and reproducible data, however, cold methanol-water quenching caused leakage and substantial loss of metabolites from various compound classes, while fast filtering and centrifugation produced quite similar metabolite pool sizes, even for metabolites with predicted high turnover. This indicates that cyanobacterial metabolic pools, as measured by GC-MS, do not show high turnover under standard growing conditions. As well, using stable (13)C labeling we show the biological origin of some of the consistently observed unknown analytes. With the development of these techniques, we establish the basis for broad scale comparative metabolite profiling of cyanobacteria.
Krall
Leonard
L
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
Huege
Jan
J
Catchpole
Gareth
G
Steinhauser
Dirk
D
Willmitzer
Lothar
L
D002247:Carbon Isotopes; D002498:Centrifugation / Q000379:methods; D016000:Cluster Analysis; D005374:Filtration / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D007553:Isotope Labeling; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D046937:Nostoc / Q000378:metabolism*; D025341:Principal Component Analysis; D015203:Reproducibility of Results; D046939:Synechocystis / Q000378:metabolism*
D002247:Carbon Isotopes