Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA333492

Local Sample ID:	S25061
Subject ID:	SU003217
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU003217
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
S25061	SA333492	FL039603	Ovarian cancer cells	Sample source
S25061	SA333492	FL039603	C	Treatment

Collection:

Collection ID:	CO003210
Collection Summary:	To generate drug-induced PARPi resistant populations, 1×106 Kuramochi drug-naïve cells (C) were initially seeded in 150 mm plates.
Sample Type:	Ovarian cancer cells

Treatment:

Treatment ID:	TR003226
Treatment Summary:	Twenty four hours after seeding, 1 uM of olaparib (Selleckchem, S1060) was added and cells were maintained under treatment until reaching confluency (> 70%), thus characterizing resistance at this dose. Cells were harvested (0.25% Trypsin/EDTA for 5 min at 37°C) and a fraction of the surviving population (1×106 cells) was seeded again and treated with 2.5 uM until cells reached confluency. This process was repeated sequentially by doubling the drug concentrations until the cell populations were able to achieve confluency at 320 uM of olaparib (T320). The initial dose was determined by cell viability assays, indicating a low starting dose (< IC30). At each step, aliquots of the adapted populations were frozen (10% DMSO, 50% FBS, 40% media) for further experiments

Treatment ID:

TR003226

Treatment Summary:

Twenty four hours after seeding, 1 uM of olaparib (Selleckchem, S1060) was added and cells were maintained under treatment until reaching confluency (> 70%), thus characterizing resistance at this dose. Cells were harvested (0.25% Trypsin/EDTA for 5 min at 37°C) and a fraction of the surviving population (1×106 cells) was seeded again and treated with 2.5 uM until cells reached confluency. This process was repeated sequentially by doubling the drug concentrations until the cell populations were able to achieve confluency at 320 uM of olaparib (T320). The initial dose was determined by cell viability assays, indicating a low starting dose (< IC30). At each step, aliquots of the adapted populations were frozen (10% DMSO, 50% FBS, 40% media) for further experiments

Sample Preparation:

Sampleprep ID:	SP003223
Sampleprep Summary:	Kuramochi cells (C and T320) were cultured until ~80% confluence either on 320 uM of olaparib for 24h or off treatment in triplicates per condition. Cell pellets were collected and frozen with liquid nitrogen for submission to the NYU Metabolomics Core Resource Laboratory. Samples were subjected to an LCMS analysis to detect and quantify known peaks. A metabolite extraction was carried out on each sample based on a previously described method116.

Sampleprep ID:

SP003223

Sampleprep Summary:

Kuramochi cells (C and T320) were cultured until ~80% confluence either on 320 uM of olaparib for 24h or off treatment in triplicates per condition. Cell pellets were collected and frozen with liquid nitrogen for submission to the NYU Metabolomics Core Resource Laboratory. Samples were subjected to an LCMS analysis to detect and quantify known peaks. A metabolite extraction was carried out on each sample based on a previously described method116.

Combined analysis:

Analysis ID	AN005076
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Ulitmate 3000
Column	SeQuant ZIC-pHILIC (150 x 4.6mm,5um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	Arbitrary units

Chromatography:

Chromatography ID:	CH003833
Methods Filename:	L12
Chromatography Comments:	The LC column was a MilliporeTM ZIC-pHILIC (2.1 x150 mm, 5 μm) coupled to a Dionex Ultimate 3000TM system and the column oven temperature was set to 25oC for the gradient elution. A flow rate of 100 μL/min was used with the following buffers; A) 10 mM ammonium carbonate in water, pH 9.0, and B) neat acetonitrile. The gradient profile was as follows; 80-20%B (0-30 min), 20-80%B (30-31 min), 80-80%B (31-42 min). Injection volume was set to 2 μL for all analyses (42 min total run time per injection).
Instrument Name:	Ulitmate 3000
Column Name:	SeQuant ZIC-pHILIC (150 x 4.6mm,5um)
Column Pressure:	1800
Column Temperature:	25
Flow Gradient:	80-20%B (0-30 min), 20-80%B (30-31 min), 80-80%B (31-42 min)
Flow Rate:	0.1mL/min
Injection Temperature:	4
Internal Standard:	ISTD (500nM amino acid cocktail)
Sample Injection:	2uL
Solvent A:	100% water; 10 mM ammonium carbonate, pH 9.0
Solvent B:	100% acetonitrile
Analytical Time:	30m
Oven Temperature:	28
Chromatography Type:	HILIC

MS:

MS ID:	MS004814
Analysis ID:	AN005076
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	M.13 Polar metabolites (pHILIC Hybrid). MS analyses were carried out by coupling the LC system to a Thermo Q Exactive HFTM mass spectrometer operating in heated electrospray ionization mode (HESI). Method duration was 30 min with a polarity switching data-dependent Top 5 method for both positive and negative modes. Spray voltage for both positive and negative modes was 3.5kV and capillary temperature was set to 320oC with a sheath gas rate of 35, aux gas of 10, and max spray current of 100 μA. The full MS scan for both polarities utilized 120,000 resolution with an AGC target of 3e6 and a maximum IT of 100 ms, and the scan range was from 67-1000 m/z. Tandem MS spectra for both positive and negative mode used a resolution of 15,000, AGC target of 1e5, maximum IT of 50 ms, isolation window of 0.4 m/z, isolation offset of 0.1 m/z, fixed first mass of 50 m/z, and 3-way multiplexed normalized collision energies (nCE) of 10, 35, 80. The minimum AGC target was 1e4 with an intensity threshold of 2e5. All data were acquired in profile mode.
Ion Mode:	UNSPECIFIED