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MB Sample ID: SA023589
Local Sample ID: | 3 |
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 |
---|---|---|---|---|
3 | SA023589 | FL006329 | 24hr | Time |
3 | SA023589 | FL006329 | Englerin (100 nM) | 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 |