van Dam, P. T. N. | bioRxiv Science

bioRxiv2026

A protocol for lab-scale production of 13C yeast extract as internal standard for metabolomics and quantification of intracellular metabolites

Metabolomics enables the profiling of small-molecule metabolites and thereby captures the biochemical state of a living organism at a given moment and enables to monitor its cellular responses to stimuli. This technique has become a powerful tool in pharmaceutical research, the food industry, and microbial research. Metabolomics aims to obtain an unbiased metabolic profile; however, this is complicated by compound instability, complex and often extensive sample processing, and nonlinear responses in mass spectrometry. Therefore, correcting for metabolite loss and mass spectrometry-related artifacts is essential, typically achieved through relative quantification against an isotopically labelled internal standard for each metabolite of interest. This article describes how to produce 13C-labelled yeast extract and its use as internal standard for metabolomics. More specifically, it provides step-by-step protocols for the fed-batch fermentation, quenching, metabolite extraction, and LC-MS and GC-MS characterization of the internal standard. It also includes a protocol explaining how to use the internal standard for the quantification of metabolites in yeast samples.

biochemistry↗

bioRxiv2022

Comparison of two sampling, quenching and extraction methods for quantitative yeasts metabolomics

Knowing intracellular metabolite concentrations is important for fundamental metabolism research as well as for biotechnology. The first steps in a quantitative metabolomics workflow, i.e., sampling, quenching, and extraction, are key to obtaining unbiased quantitative data. A qualified quenching and extraction method not only needs to rapidly terminate the in vivo biochemical reaction activities to preserve the endogenous metabolite levels but also has to fully extract all metabolites from cells. Recently, two different filtration-based sampling, quenching, and extraction methods have been proposed and used for quantitative yeast metabolomics. One method integrates the quenching and extraction into one step using a methanol-acetonitrile-water solution after a filtration step, while the other --more conventional-- method quenches with cold methanol and extracts with boiling ethanol. In this study, we tested whether these two methods are equally well suited for quantitative metabolome analyses with yeast. Using isotope dilution mass spectrometry (IDMS) with GC-MS and LC-MS as analytical methods, in combination with precise quantification of cell volumes, we determined absolute concentrations of 63 intracellular metabolites covering amino acids, organic acids, phosphorylated sugars, and nucleotides in two S. cerevisiae strains with different physiology. By analyzing the data from samples generated with the two methods, we found that while both methods yielded essentially identical concentrations for amino and organic acids, the cold-solvent extraction yielded significantly lower concentrations for particularly phosphorylated sugars and nucleotides, presumably because of lower quenching or extraction efficiency of this method. Our results show that methanol-quenching combined with boiling-ethanol-extraction is the more accurate approach when aiming to quantify a broad range of different metabolites from yeast cells. The significant discrepancy observed between both common metabolite extraction methods demonstrates the importance of method optimization for quantitative studies in particular when working with microbes with rigid cell walls such as those found in yeast.

systems biology↗