Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA239034

Local Sample ID:	u0.316_pool1_PS
Subject ID:	SU002486
Subject Type:	Yeast
Subject Species:	Saccharomyces cerevisiae
Taxonomy ID:	4932
Gender:	Not applicable

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Subject:

Subject ID:	SU002486
Subject Type:	Yeast
Subject Species:	Saccharomyces cerevisiae
Taxonomy ID:	4932
Gender:	Not applicable

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
u0.316_pool1_PS	SA239034	FL029782	Glucose	Treatment

Collection:

Collection ID:	CO002479
Collection Summary:	Cells were washed with cold PBS and them flash-frozen in liquid N2
Sample Type:	Yeast cells
Storage Conditions:	-80℃

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Treatment Summary:	For quantification of intracellular metabolite concentrations,1.0 mL-broth samples were rapidly withdrawn and quenched in pre-weighed tubes containing 5 mL cold (−40 °C) pure methanol followed immediately by vigorous vortexing.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For quantification of intracellular metabolite concentrations,1.0 mL-broth samples were rapidly withdrawn and quenched in pre-weighed tubes containing 5 mL cold (−40 °C) pure methanol followed immediately by vigorous vortexing.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.

Sample Preparation:

Sampleprep ID:	SP002492
Sampleprep Summary:	Total liquids are divided into liquid phase vials.
Processing Storage Conditions:	4℃
Extract Storage:	On ice

Combined analysis:

Analysis ID	AN003904
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters NanoAcquity
Column	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Thermo TSQ Quantum Ultra
Ion Mode	NEGATIVE
Units	umol/l

Chromatography:

Chromatography ID:	CH002890
Instrument Name:	Waters NanoAcquity
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	40
Flow Rate:	0.2ml/min
Solvent A:	100% water; 5 mM ammonium formate
Solvent B:	85% acetonitrile/15% water
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003643
Analysis ID:	AN003904
Instrument Name:	Thermo TSQ Quantum Ultra
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Xcalibur
Ion Mode:	NEGATIVE