title: Fit-for-purpose quenching and extraction protocols for metabolic profiling of yeast using chromatography-mass spectrometry platforms.
abstract: Metabolomics involves the investigation of the intracellular (endometabolome) and extracellular (exometabolome) pools of metabolites in biological systems. Methods to sample the exometabolome and to quench metabolism and extract intracellular metabolites for the model eukaryote Saccharomyces cerevisiae are presented here. These methods have been developed and validated to provide a fit-for-purpose protocol for global analyses of the S. cerevisiae metabolome. The protocol allows the extraction of a wide variety of metabolite classes and provides reproducible results to allow relative and semi-quantitative comparisons between samples of different origin. For exometabolome studies, fast sampling and separation of cells by syringe filtration is recommended. For endometabolome studies, fast quenching of intracellular metabolism is performed using a 60:40 (v/v) methanol:aqueous ammonium hydrogen carbonate solution at -48 °C. Extraction of intracellular metabolites is performed using multiple freeze/thaw cycles in a 60:40 (v/v) methanol:water solution at temperatures lower than 0 °C.
authors.affiliation: School of Chemistry, Manchester Centre for Integrative Systems Biology, Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, UK.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Winder
authors.forename: Warwick B
authors.initials: WB
authors.lastname: Dunn
mesh_terms: D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D042541:Intracellular Space / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins