title: Preparation of Intracellular Metabolite Extracts from Liquid Schizosaccharomyces pombe Cultures.
abstract: The success of metabolomic analysis relies heavily on the sample preparation protocol. Here we present a protocol for intracellular metabolite extraction from liquid fission yeast cultures based on rapid quenching in pure methanol at -40°C, bead-beating in 50% methanol for cell disruption, and 10 kDa cutoff ultrafiltration for removal of proteins. Samples are concentrated by vacuum evaporation and resuspended in 50% acetonitrile for mass spectrometric analysis. This protocol is optimal for extraction of polar metabolites such as amino acids, organic acids, nucleotides, sugars, or sugar-phosphates. Its implementation requires <6 h and allows preparation of multiple samples in parallel.
authors.affiliation: G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University (OIST), Onna-son, Kunigami, Okinawa 904-0495, Japan.
authors.forename: Tomáš
authors.identifier: 0000-0002-6940-3006
authors.initials: T
authors.lastname: Pluskal
authors.affiliation: G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University (OIST), Onna-son, Kunigami, Okinawa 904-0495, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University (OIST), Onna-son, Kunigami, Okinawa 904-0495, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Yanagida
mesh_terms: D045424:Complex Mixtures; D003470:Culture Media / Q000737:chemistry; D003890:Desiccation; D005615:Freezing; D013058:Mass Spectrometry; D055432:Metabolomics / Q000379:methods*; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000254:growth & development; D014462:Ultrafiltration
chemical_list: D045424:Complex Mixtures; D003470:Culture Media