Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA255585

Local Sample ID:	S1S2 2h_3
Subject ID:	SU002642
Subject Type:	Yeast
Subject Species:	Saccharomyces cerevisiae
Taxonomy ID:	4932
Genotype Strain:	CEN.P.K

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Subject:

Subject ID:	SU002642
Subject Type:	Yeast
Subject Species:	Saccharomyces cerevisiae
Taxonomy ID:	4932
Genotype Strain:	CEN.P.K

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
S1S2 2h_3	SA255585	FL032721	2h	label

Collection:

Collection ID:	CO002635
Collection Summary:	Care was taken to quench cells quickly in -40℃ and maintain metabolites in acid to minimize oxidation
Sample Type:	Yeast cells

Treatment:

Treatment ID:	TR002654
Treatment Summary:	Mutant SAM1SAM2 DKO cells were cultured in SD medium with the supplements of SAM and growed to log phase, then cells were washed with normal SD medium and re-cultured in SD medium

Sample Preparation:

Sampleprep ID:	SP002648
Sampleprep Summary:	Briefly, equal OD units of cells were rapidly quenched to stop metabolism by addition into 4 volumes of quenching buffer containing 60% methanol and 10 mM Tricine, pH 7.4 that was pre-cooled to -40°C. 5 min after holding at -40°C, cells were spun at 5,000 g for 3 min at 0°C, washed with the same buffer, and then resuspended in 1 mL extraction buffer containing 75% ethanol and 0.5 mM Tricine, pH 7.4. Intracellular metabolites were extracted by incubating at 75°C for 3 min, followed by chilling on ice for 5 min. Samples were spun at 20,000 g for 1 min to pellet cell debris, and 0.9 mL of the supernatant was transferred to a new tube. After a second spin at 20,000 g for 10 min, 0.8 mL of the supernatant was transferred to a new tube. Metabolites in the extraction buffer were dried using a vacuum concentrator system (Labconco) and stored at -80°C until analysis. Dried metabolite extracts were resuspended in 60% acetonitrile for injection.

Sampleprep ID:

SP002648

Sampleprep Summary:

Briefly, equal OD units of cells were rapidly quenched to stop metabolism by addition into 4 volumes of quenching buffer containing 60% methanol and 10 mM Tricine, pH 7.4 that was pre-cooled to -40°C. 5 min after holding at -40°C, cells were spun at 5,000 g for 3 min at 0°C, washed with the same buffer, and then resuspended in 1 mL extraction buffer containing 75% ethanol and 0.5 mM Tricine, pH 7.4. Intracellular metabolites were extracted by incubating at 75°C for 3 min, followed by chilling on ice for 5 min. Samples were spun at 20,000 g for 1 min to pellet cell debris, and 0.9 mL of the supernatant was transferred to a new tube. After a second spin at 20,000 g for 10 min, 0.8 mL of the supernatant was transferred to a new tube. Metabolites in the extraction buffer were dried using a vacuum concentrator system (Labconco) and stored at -80°C until analysis. Dried metabolite extracts were resuspended in 60% acetonitrile for injection.

Combined analysis:

Analysis ID	AN004188	AN004189
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Shimadzu 20AD	Shimadzu 20AD
Column	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	QTRAP	QTRAP
MS instrument name	ABI Sciex 6500+	ABI Sciex 6500+
Ion Mode	POSITIVE	NEGATIVE
Units	Peak intensity	Peak intensity

Chromatography:

Chromatography ID:	CH003103
Instrument Name:	Shimadzu 20AD
Column Name:	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:	45
Flow Gradient:	0.01 min 80% B, 20 min 20% B, 20.5 min 80% B, 34 min 80% B.
Flow Rate:	0.15ml/min
Solvent A:	20 mM ammonium carbonate and 0.1% (v/v) ammonium hydroxide
Solvent B:	acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS003935
Analysis ID:	AN004188
Instrument Name:	ABI Sciex 6500+
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	SCIEX OS
Ion Mode:	POSITIVE

MS ID:	MS003936
Analysis ID:	AN004189
Instrument Name:	ABI Sciex 6500+
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	SCIEX OS
Ion Mode:	NEGATIVE