title: Evaluation and optimization of metabolome sample preparation methods for Saccharomyces cerevisiae.
abstract: Metabolome sampling is one of the most important factors that determine the quality of metabolomics data. The main steps in metabolite sample preparation include quenching and metabolite extraction. Quenching with 60% (v/v) cold methanol at -40 °C has been most commonly used for Saccharomyces cerevisiae, and this method was recently modified as "leakage-free cold methanol quenching" using pure methanol at -40 °C. Boiling ethanol (75%, v/v) and cold pure methanol are the most widely used extraction solvents for S. cerevisiae. In the present study, metabolome sampling protocols, including the above methods, were evaluated by analyzing 110 identified intracellular metabolites of S. cerevisiae using gas chromatography/time-of-flight mass spectrometry. According to our results, fast filtration followed by washing with an appropriate volume of water can minimize the metabolite loss due to cell leakage as well as the contamination by extracellular metabolites. For metabolite extraction, acetonitrile/water mixture (1:1, v/v) at -20 °C was the most effective. These results imply that the systematic evaluation of existing methods and the development of customized methods for each microorganism are critical for metabolome sample preparation to facilitate the reliable and accurate analysis of metabolome.
authors.affiliation: School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea.
authors.forename: Sooah
authors.initials: S
authors.lastname: Kim
authors.forename: Do Yup
authors.initials: DY
authors.lastname: Lee
authors.forename: Gert
authors.initials: G
authors.lastname: Wohlgemuth
authors.forename: Hyong Seok
authors.initials: HS
authors.lastname: Park
authors.forename: Oliver
authors.initials: O
authors.lastname: Fiehn
authors.forename: Kyoung Heon
authors.initials: KH
authors.lastname: Kim
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D000431:Ethanol / Q000737:chemistry; D005374:Filtration; D008401:Gas Chromatography-Mass Spectrometry; D055442:Metabolome; D000432:Methanol / Q000737:chemistry; D025341:Principal Component Analysis; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D000097:Acetonitriles; D000431:Ethanol; D000432:Methanol; C032159:acetonitrile