A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

Citation: Sasidharan K, Soga T, Tomita M, Murray DB ( A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses Kalesh Sasidharan 0 Tomoyoshi Soga 0 Masaru Tomita 0 Douglas B. Murray 0 Mick F. Tuite, University of Kent, United Kingdom 0 Institute for Advanced Biosciences, Keio University , Nipponkoku 403-1, Daihouji, Tsuruoka City, Yamagata , Japan There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol Nethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously. - A comprehensive in vivo understanding of the underlying dynamics of metabolite reaction networks, enzyme kinetics and signalling requires the precise characterisation of intracellular metabolites at specific time points. Recent advances in highthroughput mass-spectrometry allow for the detailed metabolomewide analysis with high accuracy [14]. However, development of metabolite extraction protocols has generally lagged behind detection methods. These protocols often suffer from complex experimental design (making time-series analysis difficult), metabolite leakage during processing, metabolite oxidation during sampling/extraction [5,6], and metabolite specificity (acid-stable or alkali-stable metabolites). Furthermore, yields are influenced by the metabolic state (growth phase and rate) and properties of the species and/or strains used [710]. This leads to the paradoxical situation where the extraction protocol dictates the experimental conditions. An ideal metabolite extraction protocol should rapidly sample and quench the underlying metabolic processes, i.e., minimise degradation and modification of metabolites, and have a high and reproducible yield [7,10,11]. Turnover rates of metabolic intermediates change in the order of seconds and are highly sensitive to the changes in external conditions, thus rapid quenching is required [10,12,13]. Therefore, direct centrifugation or filtration of live cells prior to quenching should be avoided as they may alter the metabolite profile [7,10]. However, the removal and subsequent analysis of the culture media is highly desirable and must be done rapidly to avoid interfering with intracellular metabolite concentrations [5,7,10]. Furthermore, the culture media may contain a sufficiently high concentration of salts that may suppress signals or interfere with the chromatography/electrophoresis stage. There are several methods widely used for extracting metabolites from yeast cells such as freeze-thaw, sonication, hot water, boiling ethanol, permeabilisation using chloroform and treatment with extreme pH. However, these methods are mostly optimised and tested only on fast-growing low-density laboratory strains [9,1113] and the extraction buffers often use salts that are not compatible with capillary electrophoresis mass spectrometry (CEMS). Moreover, non-laboratory strains are usually nutrient limited, slow-growing and recalcitrant to lysis. This resistance arises from changes in the cell wall structure [14,15]. These physiological changes lead to differences in metabolite extraction efficiency and reproducibility, which are critical factors for the analysis of cultures grown in different conditions. A combined chemical-mechanical disruption by bead-beating using zirconia/silica beads has a high and consistent cell disruption efficiency independent of respiratory state and the cell division cycle [16]. It has also been observed that the metabolic reactions of Saccharomyces cerevisiae can be efficiently quenched in methanol (final methanol concentration .50% v/v) at 240uC and having very low metabolite leaching when the media is removed [5,12]. Moreover, extraction with chloroform-methanol has been used for the quantitative metabolite extraction of rapidly-growing, glucoserepressed laboratory strains of S. cerevisiae [9,11,12,17]. The permeabilisation technique used was time consuming (45 min) and it is known that many laboratory strains have been selected for their ability to be easily disrupted. Any metabolite modifications happening during the extraction procedure can be avoided partially by keeping a low temperature (,220uC) throughout the extraction process [10]. However, oxidation of metabolites remains an issue, for example, precise quantification of the redox sate of thiol groups are critical for understanding redox biochemistry in vivo [18]. However, oxidation of thiol groups during extraction usually hinders the accurate determination of redox state [6]. Previously we developed efficient methods for the disruption of budding yeasts for mRNA, proteins and DNA [16]. We optimised these methods for metabolite analyses that rapidly extract and fractionate the intracellular and extracellular metabolites. Our method was based on the rapid quenching of cell cultures with 280uC methanol and N-ethylmaleimide (NEM) solution. Here, NEM was used to protect thiols from oxidation by binding to -SH groups (figure 1) [19]. The quenching solution (which contains the extracellular metabolites) was rapidly removed and lyophilised. Cell pellets were bead-beated in chloroform/methanol/internal standard (IS) solution. Intracellular and extracellular metabolites were analysed using CE-MS. In parallel, we fixed a sample in ethanol to determine dry cell weight and cell number. We tested our method on continuously grown industrial S. cerevisiae cultures and it outperformed other tested extraction techniques (freezethaw and sonication), protected the redox state of the cell, has the potential to cover a large fraction of the known yeast metabolome, and gave comparable yields to those reported values for extracted metabolites from cells grown under similar conditions. Strain and Culture Conditions Unless stated otherwise, all chemicals were supplied by Wako Chemicals, Japan or Fisher Chemicals, UK. In this study we used IFO 0233 diploid strain of Saccharomyces cerevisiae. The colonies were maintained at 4uC on yeast extract peptone dextrose (YEPD) agar plates, comprising 10 g/L yeast extract (Becton Dickinson, Japan/UK), 20 g/L glucose monohydrate, 20 g/L mycolog (...truncated)