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Quenching for Microalgal Metabolomics: A Case Study on the Unicellular Eukaryotic Green Alga Chlamydomonas reinhardtii

1
Department of Chemical and Biological Engineering, ChELSI Institute, Advanced Biomanufacturing Centre, The University of Sheffield, Sheffield S1 3JD, UK
2
Department of Biosciences, College of Science, Swansea University, Swansea SA2 8PP, UK
*
Author to whom correspondence should be addressed.
Metabolites 2018, 8(4), 72; https://doi.org/10.3390/metabo8040072
Received: 14 October 2018 / Revised: 25 October 2018 / Accepted: 29 October 2018 / Published: 31 October 2018
(This article belongs to the Special Issue Metabolites from Phototrophic Prokaryotes and Algae Volume 2)
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Abstract

Capturing a valid snapshot of the metabolome requires rapid quenching of enzyme activities. This is a crucial step in order to halt the constant flux of metabolism and high turnover rate of metabolites. Quenching with cold aqueous methanol is treated as a gold standard so far, however, reliability of metabolomics data obtained is in question due to potential problems connected to leakage of intracellular metabolites. Therefore, we investigated the influence of various parameters such as quenching solvents, methanol concentration, inclusion of buffer additives, quenching time and solvent to sample ratio on intracellular metabolite leakage from Chlamydomonas reinhardtii. We measured the recovery of twelve metabolite classes using gas chromatography mass spectrometry (GC-MS) in all possible fractions and established mass balance to trace the fate of metabolites during quenching treatments. Our data demonstrate significant loss of intracellular metabolites with the use of the conventional 60% methanol, and that an increase in methanol concentration or quenching time also resulted in higher leakage. Inclusion of various buffer additives showed 70 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) to be suitable. In summary, we recommend quenching with 60% aqueous methanol supplemented with 70 mM HEPES (−40 °C) at 1:1 sample to quenching solvent ratio, as it resulted in higher recoveries for intracellular metabolites with subsequent reduction in the metabolite leakage for all metabolite classes. View Full-Text
Keywords: metabolomics; microalgae; quenching; Chlamydomonas reinhardtii; gas chromatography mass spectrometry (GC-MS) metabolomics; microalgae; quenching; Chlamydomonas reinhardtii; gas chromatography mass spectrometry (GC-MS)
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Kapoore, R.V.; Vaidyanathan, S. Quenching for Microalgal Metabolomics: A Case Study on the Unicellular Eukaryotic Green Alga Chlamydomonas reinhardtii. Metabolites 2018, 8, 72.

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