Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA239031

Local Sample ID:	u0.027_pool1_PS
Subject ID:	SU002486
Subject Type:	Yeast
Subject Species:	Saccharomyces cerevisiae
Taxonomy ID:	4932
Gender:	Not applicable

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Treatment:

Treatment ID:	TR002498
Treatment Summary:	For quantification of intracellular metabolite concentrations,1.0 mL-broth samples were rapidly withdrawn and quenched in pre-weighed tubes containing 5 mL cold (−40 °C) pure methanol followed immediately by vigorous vortexing. The quenched samples were rapidly weighed and poured into a filtration device containing a cellulose membrane and previously layered with 15 mL of cold methanol (−40 °C). Subsequently, a vacuum was applied followed by an immediate additional washing step with 15 mL cold methanol (−40 °C). The filter containing the cold washed biomass was then transferred into a 50 mL-falcon tube containing 30 mL of preheated (75 °C) aqueous ethanol solution (75% v/v). 100 μL of 13C cell extract was added to the tube as an internal standard. The tube containing the sample was then tightly closed, shaken vigorously, and placed into a water bath at 95 °C during 3 min for metabolite extraction. The tubes were then cooled using an ice bath and the filter was removed. This extract was then concentrated by complete evaporation of the ethanol–water mixture under vacuum, and resuspended in 500 μL milliQ water. After a first centrifugation at 15000 g for 5 min at 1 °C, the supernatant was transferred into a centrifugal filter unit and centrifuged again at the same conditions. The filtrate was placed into a screw-capped polypropylene vial and stored at −80 °C until further analysis. Samples were analyzed by LC-MS.

Treatment ID:

TR002498

Treatment Summary:

For quantification of intracellular metabolite concentrations,1.0 mL-broth samples were rapidly withdrawn and quenched in pre-weighed tubes containing 5 mL cold (−40 °C) pure methanol followed immediately by vigorous vortexing. The quenched samples were rapidly weighed and poured into a filtration device containing a cellulose membrane and previously layered with 15 mL of cold methanol (−40 °C). Subsequently, a vacuum was applied followed by an immediate additional washing step with 15 mL cold methanol (−40 °C). The filter containing the cold washed biomass was then transferred into a 50 mL-falcon tube containing 30 mL of preheated (75 °C) aqueous ethanol solution (75% v/v). 100 μL of 13C cell extract was added to the tube as an internal standard. The tube containing the sample was then tightly closed, shaken vigorously, and placed into a water bath at 95 °C during 3 min for metabolite extraction. The tubes were then cooled using an ice bath and the filter was removed. This extract was then concentrated by complete evaporation of the ethanol–water mixture under vacuum, and resuspended in 500 μL milliQ water. After a first centrifugation at 15000 g for 5 min at 1 °C, the supernatant was transferred into a centrifugal filter unit and centrifuged again at the same conditions. The filtrate was placed into a screw-capped polypropylene vial and stored at −80 °C until further analysis. Samples were analyzed by LC-MS.