title: A yeast metabolite extraction protocol optimised for time-series analyses.
abstract: 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 N-ethylmaleimide 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.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Nipponkoku 403-1, Daihouji, Tsuruoka City, Yamagata, Japan.
authors.forename: Kalesh
authors.initials: K
authors.lastname: Sasidharan
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Soga
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Murray
mesh_terms: D002138:Calibration; D019075:Electrophoresis, Capillary; D005033:Ethylmaleimide / Q000378:metabolism; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors
chemical_list: D013438:Sulfhydryl Compounds; D005033:Ethylmaleimide