Cyanobacteria are environmentally important photosynthetic microorganisms attracting a growing attention in various areas of basic and applied researches. To better understand their metabolism, we presently report on the development of a robust and simple protocol for facile extraction and high throughput analysis of the metabolites of the widely-used strain Synechocystis PCC6803 through liquid chromatography coupled to high resolution mass spectrometry (LC/MS). Our analytical method was developed and tested with 102 reference compounds representative of the chemical diversity of polar cell metabolites, and Synechocystis cell extracts spiked with 37 reference compounds. These samples were analyzed with two chromatographic systems, each coupled to a LTQ-Orbitrap mass spectrometer: a liquid chromatographic system equipped with a pentafluorophenylpropyl column (the PFPP-LC/MS system), and an ultra-high performance liquid chromatographic system with a C18-reversed phase column (the C18-UHPLC/MS system). We showed that the PFPP-LC/MS method performs better than the C18-UHPLC/MS method in terms of retention, separation and detection of metabolites. Consequently, we applied the PFPP-LC/MS method to analyze the metabolome of Synechocystis growing under various conditions of light and glucose, which strongly influence cell growth. We found that glucose increases glucose storage (synthesis of glycogen-like polysaccharide) and catabolism (oxidative pentose phosphate pathway and glycolysis), while it decreases the Calvin–Benson cycle that consumes photosynthetic electrons for CO2 assimilation. Depending on light and glucose availabilities, this global metabolic reprogramming can generate an oxidative stress, likely through the recombination of the glucose-spared electrons with the photosynthetic oxygen thereby producing toxic reactive oxygen species.
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Capillary electrophoresis coupled to mass spectrometry
Direct introduction mass spectrometry
Electrospray source ionization
Gas chromatography coupled to mass spectrometry
Liquid chromatography coupled to mass spectrometry
Oxidative Pentose phosphate pathway
Ultra high performance liquid chromatography
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We thank our colleagues Geoffrey Madalinski, Aurélie Roux and Ying Xu for their help with the Orbitrap machine, and Eric Ezan and Jean-François Heilier for valuable discussion. This work was supported by French agency for research Grant ANR-09-BIOE-002-01 (EngineeringH2cyano) and CNRS (Centre National recherche scientifique) Programme Interdisciplinaire Energie PIE2 (Reprogramhydrogen). KN was a recipient of PhD thesis fellowship from the CEA-Saclay (Commissariat à l’énergie atomique) France.
Christophe Junot and Franck Chauvat contributed equally to this work.
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Narainsamy, K., Cassier-Chauvat, C., Junot, C. et al. High performance analysis of the cyanobacterial metabolism via liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer: evidence that glucose reprograms the whole carbon metabolism and triggers oxidative stress. Metabolomics 9, 21–32 (2013). https://doi.org/10.1007/s11306-011-0382-4
- Central carbon metabolism
- Matrix effect
- Metabolic reprogramming