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MB Sample ID: SA023590
| Local Sample ID: | 13 |
| Subject ID: | SU000486 |
| Subject Type: | Human renal cancer cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000486 |
| Subject Type: | Human renal cancer cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 13 | SA023590 | FL006330 | 48hr | Time |
| 13 | SA023590 | FL006330 | Control | Treatment |
Collection:
| Collection ID: | CO000480 |
| Collection Summary: | Cell extracts were obtained by the addition of 1 ml of ice-cold methanol:water (80:20) to each dish followed by scraping cells into 1.7ml Eppendorf tubes and vortexing vigorously for 30 sec. A 50 ul aliquot of sample was removed for the picogreen DNA assay for purposes of biomass normalization, and the remaining sample was kept at -80˚C until ready for metabolomic analysis. |
| Sample Type: | Kidney |
Treatment:
| Treatment ID: | TR000500 |
| Treatment Summary: | A498 cells were plated in 100mm dishes at 0.5 x 106 and 1 x 106 cells/dish in complete RPMI for control and treated cells, respectively. The following day, cells were refed with complete RPMI containing 0.1% DMSO or 100 nM englerin A in DMSO with each condition being conducted in quadruplicate. Cells were incubated with vehicle or englerin A for 24 or 48 h and then they were snap frozen with liquid nitrogen. |
| Treatment: | Anticaner drug |
| Treatment Compound: | Englerin A |
| Treatment Route: | Direct incubation |
| Treatment Dose: | 100 nM |
| Treatment Doseduration: | 24hr and 48hr |
Sample Preparation:
| Sampleprep ID: | SP000493 |
| Sampleprep Summary: | Typically, 300 µl of cells in methanol:water (80:20) was thawed on ice and transferred to a 1.7 ml Eppendorf tube. Five µl of a cocktail containing 25-35 commercial stable isotope internal standards, and 5.0 µl of 57 stable isotope internal standards that were custom-synthesized in E. coli and S. cerevisiae by metabolic labeling with 13C-glucose, and 13C-bicarbonate, were added, and vortexed vigorously for 30 sec. Macromolecules (protein, DNA, RNA, glycans, etc.) then were removed by centrifugation at 16,000g x 10 min at 4˚C. The supernatants containing the extracted metabolites and internal standards were transferred to labeled cryotubes and stored at -80˚C for LC-MS/MS analysis. |
| Extract Storage: | -80C |
| Sample Derivatization: | No |
| Sample Spiking: | Five µl of a cocktail containing 25-35 commercial stable isotope internal standards, and 5.0 µl of 57 stable isotope internal standards that were custom-synthesized in E. coli and S. cerevisiae by metabolic labeling with 13C-glucose, and 13C-bicarbonate, were added |
| Cell Type: | Renal cancer cell |
Combined analysis:
| Analysis ID | AN000726 | AN000727 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Shimadzu 20AD | Shimadzu 20AD |
| Column | Luna NH2 aminopropyl HPLC | Luna NH2 aminopropyl HPLC |
| MS Type | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole |
| MS instrument name | ABI Sciex 5500 QTrap | ABI Sciex 5500 QTrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | AUC | AUC |
Chromatography:
| Chromatography ID: | CH000520 |
| Chromatography Summary: | HILIC |
| Instrument Name: | Shimadzu 20AD |
| Column Name: | Luna NH2 aminopropyl HPLC |
| Column Temperature: | 25C |
| Flow Gradient: | 0 min-95% B, 4 min-B, 19 min-2% B, 22 min-2% B, 23 min-95% B, 28 min-end. |
| Flow Rate: | 0.3 ml/min |
| Solvent A: | 95% water; 23.18mM NH4OH+20 mM formic acid (Ph 9.4) |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 28 min |
| Oven Temperature: | 25C |
| Target Sample Temperature: | 4C |
| Sample Loop Size: | 10 ul |
| Sample Syringe Size: | 100 ul |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS000643 |
| Analysis ID: | AN000726 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
| Collision Energy: | Compound-specific |
| Collision Gas: | High |
| Fragmentation Method: | MRM |
| Ion Source Temperature: | 500C |
| Ionization: | ESI |
| Mass Accuracy: | 0.1Da |
| MS ID: | MS000644 |
| Analysis ID: | AN000727 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Collision Energy: | Compound-specific |
| Collision Gas: | High |
| Fragmentation Method: | MRM |
| Ion Source Temperature: | 500C |
| Ionization: | ESI |
| Mass Accuracy: | 0.1Da |
