Targeted metabolomics profiling data (i.e., peak intensity measurements) after acute exposure of BT-549 cells to DMSO or rapamycin, AZD8055 or Torin2 at two doses (100nM, 1000nM) for 6 hours. - Dataset (ID:20368)
| HMS Dataset ID: | 20368 |
| Dataset Title: | Targeted metabolomics profiling data (i.e., peak intensity measurements) after acute exposure of BT-549 cells to DMSO or rapamycin, AZD8055 or Torin2 at two doses (100nM, 1000nM) for 6 hours. |
| Screening Lab Investigator: | Sameer Chopra |
| Screening Principal Investigator: | Peter K. Sorger |
| Assay Description: | Recovery and measurement of intracellular polar metabolites from drug-treated breast cancer cells. |
| Assay Protocol: |
1. BT-549 cells in complete media were plated in 8 x 10cm2 dishes at 2.5x106 cells per dish and cultured at 37°C and 5% CO2. Approximately two days later when cells were 75-80% confluent, the media in each dish was replaced with 10mL of fresh media containing 100nM or 1000nM of rapamycin, AZD8055 or Torin2, or an equivalent volume of DMSO. Two independent DMSO plates were prepared for each of three experimental replicates (N=8 total samples per replicate). After 6 hours of treatment, the 10cm2 dishes were placed on dry ice and the media was aspirated and replaced with ice cold 80% (v/v) methanol. Dishes were kept at -80°C for 20 minutes prior to using a cell scraper to produce a cell lysate/methanol mixture. 2. The mixture for each sample was collected in a 15mL Eppendorf tube and cellular debris and protein were pelleted by centrifugation at 1,800 x g for 5 minutes at 4°C. The methanol supernatant containing polar metabolites was transferred to a new 15mL tube on dry ice and the cell debris/protein pellets were resuspended two additional times with methanol, centrifuged and re-pelleted. Recovered methanol supernatants for each sample were pooled and then divided equally into four 1.5mL tubes on dry ice. Methanol was evaporated using a SpeedVac (no applied heat) and the resulting polar metabolite pellets were stored at -80°C. 3. For mass spectrometry, metabolites were re-suspended in 20mL HPLC-grade water for mass spectrometry. 5-7μL were injected and analyzed using a hybrid 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFPLC system (Shimadzu) via selected reaction monitoring (SRM) of a total of 262 endogenous water-soluble metabolites for steady-state analyses of samples. Some metabolites were targeted in both positive and negative ion mode for a total of 298 SRM transitions using positive/negative ion polarity switching. ESI voltage was +4950V in positive ion mode and –4500V in negative ion mode. The dwell time was 3 milliseconds per SRM transition, and the total cycle time was 1.55 seconds. Approximately 10-14 data points were acquired per detected metabolite. 4. Samples were delivered to the mass spectrometer via hydrophilic interaction chromatography (HILIC) using a 4.6mm i.d x 10cm Amide XBridge column (Waters) at 400 μL/min. Buffer A was comprised of 20mM ammonium hydroxide/20mM ammonium acetate (pH=9.0) in 95:5 water:acetonitrile and Buffer B was HPLC grade acetonitrile. Gradients were run starting from 85% buffer B (in buffer A) to 42% B from 0-5min; 42% B to 0% B from 5-16min; 0% B was held from 16-24min; 0% B to 85% B from 24-25min; 85% B was held for 7min to re-equilibrate the column. 5. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.1 software (AB/SCIEX). The accompanying data represent samples from three independent experimental replicates that were analyzed concurrently by mass spectrometry. |
| Assay Protocol Reference: | Yuan M, Breitkopf SB, Yang X, Asara JM. A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat Protoc. 2012 Apr 12;7(5):872-81. |
| HMS Dataset Type: | Metabolomics |
| Date Publicly Available: | 2019-11-12 |
| Most Recent Update: | 2019-11-12 |