Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA343200

Local Sample ID:	QC-2
Subject ID:	SU003293
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU003293
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
QC-2	SA343200	FL040567	cultured cells	Sample source
QC-2	SA343200	FL040567	QC	Factor

Collection:

Collection ID:	CO003286
Collection Summary:	Care was taken to quench cells quickly to minimize oxidation and degradation.
Sample Type:	in vitro reaction

Treatment:

Treatment ID:	TR003302
Treatment Summary:	Stress granules purified from HeLa cells were treated with 13C-Glucose for 0, 1, 3 hr reaction.

Sample Preparation:

Sampleprep ID:	SP003300
Sampleprep Summary:	Cells without stress treatment or treated with 0.6 M sorbitol stress for 1.5 h were scraped off and centrifuged to collect cell pellets. The cell pellets were thawed on ice, resuspended in stress granule lysis buffer (50 mM Tris, 100 mM K2OAc, 2 mM MgOAc, 0.5 mM DTT, 50 μg/mL heparin, 0.5% NP-40, 0.02% antifoam B, proteinase inhibitor cocktail, pH 7.4) and lysis by syringe. Lysates were centrifuged at 1000 × g for 5 min to collect supernatants. Supernatants were centrifuge at 18000 × g for 20 min to collect pellets and the pellets were washed once with stress granule lysis buffer. Then, pellets were resuspended in stress granule lysis buffer and centrifuged at 850 × g for 2 min to collect supernatants containing stress granule. Then NAD+, ATP and 13C-glucose were added for reaction at 37 'C for 0, 1 and 3 hr. The reaction was quenched by 3-fold methanol and centrifuge to collect the supernant for analysis.

Sampleprep ID:

SP003300

Sampleprep Summary:

Cells without stress treatment or treated with 0.6 M sorbitol stress for 1.5 h were scraped off and centrifuged to collect cell pellets. The cell pellets were thawed on ice, resuspended in stress granule lysis buffer (50 mM Tris, 100 mM K2OAc, 2 mM MgOAc, 0.5 mM DTT, 50 μg/mL heparin, 0.5% NP-40, 0.02% antifoam B, proteinase inhibitor cocktail, pH 7.4) and lysis by syringe. Lysates were centrifuged at 1000 × g for 5 min to collect supernatants. Supernatants were centrifuge at 18000 × g for 20 min to collect pellets and the pellets were washed once with stress granule lysis buffer. Then, pellets were resuspended in stress granule lysis buffer and centrifuged at 850 × g for 2 min to collect supernatants containing stress granule. Then NAD+, ATP and 13C-glucose were added for reaction at 37 'C for 0, 1 and 3 hr. The reaction was quenched by 3-fold methanol and centrifuge to collect the supernant for analysis.

Combined analysis:

Analysis ID	AN005209	AN005210
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Shimadzu 20AD	Shimadzu 20AD
Column	Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)	Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	QTRAP	QTRAP
MS instrument name	ABI Sciex Triple Quad 5500+	ABI Sciex Triple Quad 5500+
Ion Mode	POSITIVE	NEGATIVE
Units	Peak intensity	Peak intensity

Chromatography:

Chromatography ID:	CH003941
Instrument Name:	Shimadzu 20AD
Column Name:	Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
Column Temperature:	45
Flow Gradient:	0.01 min 80% B, 20 min 20% B, 20.5 min 80% B, 24 min 80% B.
Flow Rate:	0.15 mL/min
Solvent A:	100% water; 20 mM ammonium carbonate
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS004942
Analysis ID:	AN005209
Instrument Name:	ABI Sciex Triple Quad 5500+
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	The raw data were extracted with the software Analyst v1.7.2 and OS v1.7. This is a continuation of study ST002927 where a different set precusror/product ions were chosen for MS.
Ion Mode:	POSITIVE

MS ID:	MS004943
Analysis ID:	AN005210
Instrument Name:	ABI Sciex Triple Quad 5500+
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	The raw data were extracted with the software Analyst v1.7.2 and OS v1.7. This is a continuation of study ST002927 where a different set precursor/product ions were chosen for MS.
Ion Mode:	NEGATIVE