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

Rahul Kapoore,Seetharaman Vaidyanathan

doi:10.3390/metabo8040072

Rahul Kapoore, Seetharaman Vaidyanathan

Open Access

https://doi.org/10.3390/metabo8040072

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Journal: Metabolites	Publication Date: Oct 31, 2018
Citations: 5	License type: CC BY 4.0

Affiliation: University of Sheffield, Swansea University

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.

Highlights

The major bottlenecks associated with sample preparation include efficient sampling, quenching, and extraction of metabolites, achieved with minimal alteration of the internal metabolome signature.In order to retain a valid snapshot of the metabolome, rapid sampling and quenching of enzyme activities is a crucial step in any metabolomics workflow
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
In most of the studies that evaluated the quenching methods for bacterial models [13,17], conclusions were drawn primarily based on adenosine triphosphate (ATP) assay and adenylate energy charge

Summary

Introduction

The major bottlenecks associated with sample preparation include efficient sampling, quenching, and extraction of metabolites, achieved with minimal alteration of the internal metabolome signature.In order to retain a valid snapshot of the metabolome, rapid sampling and quenching of enzyme activities is a crucial step in any metabolomics workflow. Quenching solvent should halt the constant flux of metabolism and high turnover rate of metabolites without causing any damage to the cell membrane/wall thereby avoiding any leakage of intracellular metabolites [1,2]. Quenching with 60% v/v cold methanol at −40 ◦ C or −50 ◦ C has been used widely in the past for microbial, fungi, yeast and plant metabolomics. Potential problems connected to the leakage of intracellular metabolites with cold methanol quenching was reported later for yeast [3]. Various alternatives to cold methanol quenching, such as filter culture methodology [8], fast filtration [4], mass balance approach [9] and use of alternative quenching solvents (such as glycerol-saline, methanol/glycerol and methanol/NaCl) have been evaluated for bacterial

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Conclusion