Fast sampling for quantitative microbial metabolomics: new aspects on cold methanol quenching: metabolite co-precipitation

Abstract

The intra-and extracellular concentrations of 16 metabolites were measured in chemostat (D = 0.1 h -1 ) anaerobic cultures of the yeast Saccharomyces cerevisiae CEN.PK-113-7D growing on minimal medium. Two independent sampling workflows were employed: (i) conventional cold methanol quenching and (ii) a differential approach. Metabolites were quantified in different sample fractions (total, extracellular, quenching supernatant, methanol/water extract and pellet) in order to derive their mass balance. The differential method in combination with absolute metabolite quantification by gas-chromatography with isotope dilution mass spectrometry (GC-IDMS) was used as a benchmark to assess quality of the cold methanol quenching procedure. Quantitative comparison of metabolite concentrations in all fractions collected by different quenching techniques indicates asystematic loss of the total mass of various metabolites in course of the cold methanol quenching. Pellet resulting from the cold methanol quenching besides biomass contains considerable amounts of precipitated inorganic salts from the fermentation media. Quantitative analysis has revealed significant co-precipitation of polar extracellular metabolites together with these salts. This phenomenon is especially significant for metabolites with large extracellular mass-fraction. We report that the co-precipitation is a hitherto neglected phenomenon and concluded that its degree strongly linked to culturing conditions (i.e. media composition) and chemical properties of the particular metabolite. Thus, intracellular metabolite levels measured from samples collected by cold methanol quenching might be uncertain and variably biased due to corruption by described phenomena.

Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems

“…Further, even with a strong focus on sampling and sample processing in the field, and many available publications [25][26][27], we are still left with compromised protocols for most biological model systems [28]. Even the long-time golden standard protocol for sampling yeast in cold methanol has been re-investigated and reported sensitive to a co-precipitation phenomenon, compromising its quantitative accuracy [29]. Another challenge in the field of metabolomics is the varying physicochemical properties of the metabolite classes, and the fact that some classes are extremely labile at certain conditions.…”

Rapid sample processing for intracellular metabolite studies in Penicillium ochrochloron CBS 123.824: the FiltRes-device combines cold filtration of methanol quenched biomass with resuspension in extraction solution

“…Nielsen and Jewett 2007 ; van Gulik et al 2013 ; Villas-Bôas et al 2005 ). To complicate matters further, this methodological adaptation might even be necessary on a phenotypic level since changes in physiology, plasma membrane and cell wall composition can affect the organism’s response to the applied methods (da Luz et al 2014 ; van Gulik et al 2013 ; Zakhartsev et al 2015 ). These findings led to a series of critical evaluations and improvements of techniques for rapid sampling, quenching, separation of biomass, extraction of metabolites and evaporation (e.g.…”

Coupled metabolic‐hydrodynamic modeling enabling rational scale‐up of industrial bioprocesses

“…Nonetheless, it has been reported that conventional centrifugation‐based quenching and washing methods gave rise to serious metabolite leakage and/or cold shock during sampling process in bacteria, yeast, and filamentous fungi (Bolten, Kiefer, Letisse, Portais, & Wittmann, ; Canelas, Ras et al, ; de Jonge, Douma, Heijnen, & van Gulik, ; S. Kim et al, ; Wellerdiek, Winterhoff, Reule, Brandner, & Oldiges, ; Wittmann, Kromer, Kiefer, Binz, & Heinzle, ). Besides metabolite leakage during the quenching, incomplete removal of supernatant from cell pellet/cake and metabolite coprecipitation will aggravate the misestimation of the intracellular metabolite contents (Zakhartsev, Vielhauer, Horn, Yang, & Reuss, ). Several strategies have been advocated to account for this bias: (a) preserve cell membrane integrity by supplementing cryoprotective, pH‐stabilizing agents or osmoprotective in the quenching solution (Link, Anselment, & Weuster‐Botz, ; Schadel, David, & Franco‐Lara, ); (b) quantify and correct leakage by accounting for the sample loss in the quenching supernatant after separation (Tillack, Paczia, Noh, Wiechert, & Noack, ); (c) quench and extract the entire metabolome in a biological sample simultaneously (“whole‐broth sampling”; Canelas, Ras et al, ; Taymaz‐Nikerel et al, ).…”