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MB Sample ID: SA212944
Local Sample ID: | 109757 |
Subject ID: | SU002320 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|---|
109757 | SA212944 | FL025972 | WT | Genotype |
109757 | SA212944 | FL025972 | Etop-0h | 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. |
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. |
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 |