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.

https://www.biorxiv.org/content/10.1101/2022.02.21.481278v1.abstract@en

We here explain step by step the implementation of gas chromatography coupled with tandem mass spectrometry for the quantitative analysis of intracellular metabolites from the tricarboxylic acid (TCA) cycle such as citrate, isocitrate, alpha-ketoglutarate, succinate, malate, and fumarate. Isotope dilution is used to correct for potential metabolite losses during sample processing, matrix effects, incomplete derivatization, and liner contamination. All measurements are performed in selected reaction monitoring (SRM) mode. Standards and samples are first diluted with a fixed volume of a mixture of fully ¹³C-labeled internal standards and then derivatized to give trimethylsilyl-methoxylamine derivatives prior GC-MS/MS analysis.

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Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled 13C-cell suspension, of an E. coli PHB producing strain, grown on U-13C-glucose as C-source. This internal 13C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/gCDW) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps@en

Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of 13C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS-type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC-MS(/MS) requires derivatization for the usually non-volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full useof LC- and GC-MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; 13C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation. Biotechnol.@en