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MB Sample ID: SA309453
| Local Sample ID: | NN-022 |
| Subject ID: | SU002976 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002976 |
| 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 |
|---|---|---|---|---|
| NN-022 | SA309453 | FL037205 | MYC_Enza_C13Gln | Treatment |
Collection:
| Collection ID: | CO002969 |
| Collection Summary: | Media was aspirated and cells were washed with cold 150mM ammonium acetate pH 7.3. Metabolite extractions were performed by adding 500μl of cold 80% methanol containing 2nM Norvaline (Sigma) as an internal standard per well. Cells were removed using a cell scraper before transferring cell suspensions to 1.5ml Eppendorf tubes. Samples were vortexed for 30 seconds and spun at 4°C for 5 minutes at maximum speed to pellet the insoluble fraction before 420 µl of the soluble fraction was transferred to ABC vials (Thermo Fisher Scientific). 80% MeOH was evaporated from the ABC vials using the EZ-2Elite evaporator (Genevac) and samples were stored at -80°C until analysis. |
| Sample Type: | Prostate cancer cells |
Treatment:
| Treatment ID: | TR002985 |
| Treatment Summary: | Tissue culture plates were coated with 0.01% (v/v) Poly-L-Lysine (Sigma, P4832) diluted 1/20 in distilled water and washed with PBS to enhance cell attachment. 16D cells were cultured in RPMI base media (Gibco) + 10% FBS (v/v) + 100 units/mL penicillin, and 100μg/mL streptomycin. Enzalutamide treatment was performed by adding 10μM Enzalutamide (Selleck Chemicals, S1250) every 48 hours. |
| Treatment Compound: | Enzalutamide |
| Treatment Vehicle: | DMSO |
Sample Preparation:
| Sampleprep ID: | SP002982 |
| Sampleprep Summary: | For metabolite extraction, media was aspirated and cells were washed with cold 150mM ammonium acetate pH 7.3. Metabolite extractions were performed by adding 500μl of cold 80% methanol containing 2nM Norvaline (Sigma) as an internal standard per well. Cells were removed using a cell scraper before transferring cell suspensions to 1.5ml Eppendorf tubes. Samples were vortexed for 30 seconds and spun at 4°C for 5 minutes at maximum speed to pellet the insoluble fraction before 420 µl of the soluble fraction was transferred to ABC vials (Thermo Fisher Scientific). 80% MeOH was evaporated from the ABC vials using the EZ-2Elite evaporator (Genevac) and samples were stored at -80°C until analysis. |
Combined analysis:
| Analysis ID | AN004696 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | Phenomenex Luna NH2 (150 x 2mm,3um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Abundance |
Chromatography:
| Chromatography ID: | CH003536 |
| Chromatography Summary: | Dried metabolites were resuspended in 50% ACN:water and 1/10th was loaded onto a Luna 3um NH2 100A (150 × 2.0 mm) column (Phenomenex). The chromatographic separation was performed on a Vanquish Flex (Thermo Fisher Scientific) with mobile phases A (5 mM NH4AcO pH 9.9) and B (ACN) and a flow rate of 200 μl/minute. A linear gradient from 15% A to 95% A over 18 minutes was followed by 9 minutes isocratic flow at 95% A and reequilibration to 15% A. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Luna NH2 (150 x 2mm,3um) |
| Column Temperature: | 35 |
| Flow Gradient: | Linear gradient was as follows: 15% A to 95% A over 18 minutes was followed by 9 minutes isocratic flow at 95% A and reequilibration to 15% A |
| Flow Rate: | 200 ul/minute |
| Solvent A: | 5 mM NH4AcO pH 9.9 |
| Solvent B: | ACN |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004443 |
| Analysis ID: | AN004696 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Metabolites were detection with a Thermo Fisher Scientific Q Exactive mass spectrometer run with polarity switching (+3.5 kV/− 3.5 kV) in full scan mode with an m/z range of 70-975 and 70.000 resolution. TraceFinder 4.1 (Thermo Fisher Scientific) was used to quantify the targeted metabolites by area under the curve using expected retention time and accurate mass measurements (< 5 ppm). |
| Ion Mode: | UNSPECIFIED |
