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

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.

13C-Isotope-Assisted Assessment of Metabolic Quenching During Sample Collection from Suspension Cell Cultures

“…Alternatively, the spent medium can be initially removed by centrifugation or rapid filtration prior to freezing or quenching in cold methanol, hot methanol or ethanol, or perchloric acid. 6,9,10 Prior studies have compared an array of quenching methods for metabolomics analysis of bacteria, 8,11,12 yeast, 12,13 algae, 14 and mammalian cells, 6 but the optimal method was often found to be metabolite-and organism-specific. Therefore, refining and expanding upon current methods to assess metabolic quenching efficiency is expected to facilitate the application of metabolomics to new organisms and experimental systems.…”

“…Commonly used metabolic quenching methods include directly mixing cell culture samples with a quenching solution selected from a variety of possible options, such as cold methanol solutions, , cold media, or cold saline ice slurries. − Once quenched, the cell biomass can be physically separated from the spent medium and quenching solution prior to freezing or immediate metabolite extraction. Alternatively, the spent medium can be initially removed by centrifugation or rapid filtration prior to freezing or quenching in cold methanol, hot methanol or ethanol, or perchloric acid. ,, Prior studies have compared an array of quenching methods for metabolomics analysis of bacteria, ,, yeast, , algae, and mammalian cells, but the optimal method was often found to be metabolite- and organism-specific. Therefore, refining and expanding upon current methods to assess metabolic quenching efficiency is expected to facilitate the application of metabolomics to new organisms and experimental systems.…”

“…To date, the assessment of metabolic quenching methods has centered around evaluating several criteria such as (i) leakage of metabolites due to loss of cell integrity during quenching, , (ii) recovery of certain rapidly turned over intracellular metabolites, ,,,, (iii) breadth of detectable metabolites, , and (iv) abundance of metabolites recovered in cell extracts. ,,, However, these criteria do not directly reflect the extent of metabolite turnover that occurs during sample removal and quenching, which may mislead researchers to adopt protocols that cause dramatic shifts in metabolite concentrations or isotopic labeling prior to measurement and thereby lead to invalid conclusions. In a recent study aimed at comparing the metabolic quenching efficiency of different sampling protocols, researchers injected 13 C-bicarbonate into cultures of the cyanobacterium Synechococcus sp.…”

The Effects of Sampling and Storage Conditions on the Metabolite Profile of the Marine Sponge Geodia barretti

“…Sample collection for metabolomics strive to capture the metabolic state of the sample at the time of sampling and therefore the it should alter the metabolome as little as possible as well as quench the metabolism as quickly as possible to avoid metabolite degradation post-sampling. Furthermore, factors such as sampling, quenching the metabolism, storage conditions as well as sample extraction parameters have great effects on the final metabolome as well as data quality Engskog et al (2016) , Vuckovic (2018) , and have been investigated and reviewed for plasma, serum and urine Bi et al (2020) , González-Domínguez et al (2020) , Smith et al (2020) , mammalian cells Dettmer et al (2011) , Bi et al (2013) , Engskog et al (2015) , bacteria and microbes Mashego et al (2007) , Rabinowitz (2007) , Kapoore and Vaidyanathan (2018) , as well as for plants ( Kim and Verpoorte, 2010 ; Rodrigues et al, 2019 ; Salem et al, 2020 ). For the collection of sponges for metabolomics, no guidelines or practical consensus exist and the procedures for sampling varies, in some cases the sponges are frozen directly upon collection, in some cases the time between collection and freezing is not stated in the publications and in some cases the sponges are immediately stored in ethanol where after the ethanol is often discarded prior to extraction of the sponge ( Olsen et al, 2016b ; Einarsdottir et al, 2017 ; Bayona et al, 2018 ; Bayona et al, 2020 ; Reverter et al, 2018 ; Mohanty et al, 2020 ).…”

Mathematical modelling of cellulase production and continuous production of enzymes under carbon-limited conditions by Trichoderma harzianum P49P11

“…However, Bolten et al (2007) reported potential problems connected to the leakage of intracellular metabolites with cold methanol quenching. Kapoore and Vaidyanathan (2018) investigated the influence of various parameters such as quenching solvents, methanol concentrations and inclusion of buffer additives on intracellular metabolite leakage from Chlamydomonas reinhardtii. They reported a significant loss of intracellular metabolites with the use of the conventional 60 % (v/v) methanol, and they recommended the supplementation of 70 mM HEPES to reduce the leakage of metabolites.…”