Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA212936

Local Sample ID:	109781
Subject ID:	SU002320
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002320
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
109781	SA212936	FL025969	PRDX1 KO	Genotype
109781	SA212936	FL025969	Etop-24h	Treatment

Collection:

Collection ID:	CO002313
Collection Summary:	For metabolite collection, plates containing 0.2-0.4 million cells per well were gently washed with 75mM ammonium carbonate buffer pH 7.4 at room temperature, transferred on ice, and metabolites were extracted with 80:20 ice-cold MeOH:H2O solution. Cells were scraped off and samples were collected in tubes, then snap frozen in liquid nitrogen to stop all metabolic reactions.
Sample Type:	U2OS cells

Treatment:

Treatment ID:	TR002332
Treatment Summary:	U2-OS cells were seeded in 6 well plates. Etoposide treatment (1μM for 3 hours) was performed at different times to be able to terminate the experiment and extract the metabolites simultaneously for all samples. At the last timepoint – treatment for the no release samples – the medium was changed in all wells in order to have growth medium of the same composition at the time of metabolite extraction. Each sample was prepared in triplicates.

Treatment ID:

TR002332

Treatment Summary:

U2-OS cells were seeded in 6 well plates. Etoposide treatment (1μM for 3 hours) was performed at different times to be able to terminate the experiment and extract the metabolites simultaneously for all samples. At the last timepoint – treatment for the no release samples – the medium was changed in all wells in order to have growth medium of the same composition at the time of metabolite extraction. Each sample was prepared in triplicates.

Sample Preparation:

Sampleprep ID:	SP002326
Sampleprep Summary:	Once all wells have been collected, samples were thawed and centrifuged in a table-top centrifuge at maximum speed at 4 degrees. Supernatants containing metabolites were transferred into HPLC vial and stored at -80 degrees until processing by the metabolomics facility (Pro-Met, CeMM). Cleared extracts were dried under nitrogen. Samples were taken up in MS-grade water and mixed with the heavy isotope labeled internal standard mix.

Sampleprep ID:

SP002326

Sampleprep Summary:

Once all wells have been collected, samples were thawed and centrifuged in a table-top centrifuge at maximum speed at 4 degrees. Supernatants containing metabolites were transferred into HPLC vial and stored at -80 degrees until processing by the metabolomics facility (Pro-Met, CeMM). Cleared extracts were dried under nitrogen. Samples were taken up in MS-grade water and mixed with the heavy isotope labeled internal standard mix.

Combined analysis:

Analysis ID	AN003644
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity II
Column	Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Agilent 6470 TQ
Ion Mode	NEGATIVE
Units	au

Chromatography:

Chromatography ID:	CH002698
Chromatography Summary:	A 1290 Infinity II UHPLC system (Agilent Technologies) coupled with a 6470 triple quadrupole mass spectrometer (Agilent Technologies) was used for the LC-MS/MS analysis. The chromatographic separation for samples was carried out on a ZORBAX RRHD Extend-C18, 2.1 x 150 mm, 1.8 µm analytical column (Agilent Technologies). The column was maintained at a temperature of 40°C and 4 µL of sample was injected per run. The mobile phase A was 3% methanol (v/v), 10 mM tributylamine, 15 mM acetic acid in water and mobile phase B was 10 mM tributylamine, 15 mM acetic acid in methanol. The gradient elution with a flow rate of 0.25 mL/min was performed for a total time of 24 min. Afterwards back-flushing of the column using a 6port/2-position divert valve was carried out for 8 min using acetonitrile, followed by 8 min of column equilibration with 100% mobile phase A. The triple quadrupole mass spectrometer was operated in negative electrospray ionization mode, spray voltage 2 kV, gas temperature 150 °C, gas flow 1.3 L/min, nebulizer 45 psi, sheath gas temperature 325 °C, sheath gas flow 12 L/min. The metabolites of interest were detected using a dynamic MRM mode.
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)
Column Temperature:	40
Flow Gradient:	The gradient elution with a flow rate of 0.25 mL/min was performed for a total time of 24 mi
Flow Rate:	0.25ml/min
Solvent A:	3% methanol/97% water; 15 mM acetic acid; 10 mM tributylamine
Solvent B:	100% methanol; 15 mM acetic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003395
Analysis ID:	AN003644
Instrument Name:	Agilent 6470 TQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The chromatographic separation for samples was carried out on a ZORBAX RRHD Extend-C18, 2.1 x 150 mm, 1.8 µm analytical column (Agilent Technologies). The column was maintained at a temperature of 40°C and 4 µL of sample was injected per run. The mobile phase A was 3% methanol (v/v), 10 mM tributylamine, 15 mM acetic acid in water and mobile phase B was 10 mM tributylamine, 15 mM acetic acid in methanol. The gradient elution with a flow rate of 0.25 mL/min was performed for a total time of 24 min. Afterwards back-flushing of the column using a 6port/2-position divert valve was carried out for 8 min using acetonitrile, followed by 8 min of column equilibration with 100% mobile phase A. The triple quadrupole mass spectrometer was operated in negative electrospray ionization mode, spray voltage 2 kV, gas temperature 150 °C, gas flow 1.3 L/min, nebulizer 45 psi, sheath gas temperature 325 °C, sheath gas flow 12 L/min. The metabolites of interest were detected using a dynamic MRM mode.
Ion Mode:	NEGATIVE