Quantitative Evaluation of Intracellular Metabolite Extraction Techniques for Yeast Metabolomics

Abstract

Accurate determination of intracellular metabolite levels requires well-validated procedures for sampling and sample treatment. Several methods exist for metabolite extraction, but the literature is contradictory regarding the adequacy and performance of each technique. Using a strictly quantitative approach, we have re-evaluated five methods (hot water, HW; boiling ethanol, BE; chloroform-methanol, CM; freezing-thawing in methanol, FTM; acidic acetonitrile-methanol, AANM) for the extraction of 44 intracellular metabolites (phosphorylated intermediates, amino acids, organic acids, nucleotides) from S. cerevisiae cells. Two culture modes were investigated (batch and chemostat) to check for growth condition dependency, and three targeted platforms were employed (two LC-MS and one GC/MS) to exclude analytical bias. Additionally, for the determination of metabolite recoveries, we applied a novel approach based on addition of (13)C-labeled internal standards at different stages of sample processing. We found that the choice of extraction method can drastically affect measured metabolite levels, to an extent that for some metabolites even the direction of changes between growth conditions can be inverted. The best performances, in terms of efficacy and metabolite recoveries, were achieved with BE and CM, which yielded nearly identical levels for the metabolites analyzed. According to our results, AANM performs poorly in yeast and FTM cannot be considered adequate as an extraction method, as it does not ensure inactivation of enzymatic activity.

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry

“…Recoveries were above 87% whether standards were added before or after extraction. By contrast with studies of yeast or bacteria (Canelas et al, 2009), no quantitative evaluation of intracellular metabolite extraction techniques in plant tissues appears to have been published. When tested on E. coli and Saccharomyces cerevisiae and compared to other extraction methods (boiling ethanol, perchloric acid, or potassium hydroxide), the recoveries of intracellular metabolites after hot water extraction were excellent (Hans et al, 2001).…”

Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains

“…Samples for metabolomics were withdrawn using a custom-made low dead-volume rapid sampling setup 30 . Replicate samples were processed according to the standard procedures in use at each laboratory, resulting in seven protocol variations, although all involved cold methanol quenching 31 and boiling ethanol extraction 32 as the key steps, followed by evaporation to dryness before distribution. Samples for transcriptomics were collected by overpressure and quenched in liquid nitrogen.…”

13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea

“…For the samples (in section ''Performance of determination methods of intracellular metabolites'') used to validate the determination method, 13 C-labeled IS was added into each sample just before LC-MS/MS detection, which will not offset the losses during sample treatment but only correct for analytical artifacts (e.g., sample matrix effects). For the samples (in sections ''Metabolomics analysis of an erythromycin production process'' and ''Corroboration of the positive correlation between propionyl-CoA pool size and specific erythromycin production rate'') used to determine the intracellular metabolites concentrations, 13 Clabeled IS was added right after quenching, which compensates the losses during sample treatment (e.g., volume losses, partial degradation) [15].…”