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MB Sample ID: SA080698
Local Sample ID: | 13RWPE1_12-1 |
Subject ID: | SU001232 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001232 |
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 |
---|---|---|---|---|
13RWPE1_12-1 | SA080698 | FL012343 | RWPE1 | Cell lines |
13RWPE1_12-1 | SA080698 | FL012343 | 12h | Time points |
Collection:
Collection ID: | CO001226 |
Collection Summary: | cells were grown in RPMI 1640 medium containing 2 g/L glucose with 10% dFBS, and were harvested at 80% confluence. One hour before the isotope switch, the medium was aspirated and replaced with fresh unlabeled medium[1]. Subsequently, VCaP and RWPE-1 were cultured in RPMI 1640 medium with 2 g/L [U−13C]-glucose and 10% dFBS for 0 h, 0.25 h, 4 h, 12 h, 24 h and long-term for 6 days performing thereby 3 passages. 10 cm petri dish with 10 mL of media was used for each sample, and 4 independent biological replicates were prepared for the labeling experiments. Unlabeled samples were treated in the same way with medium containing unlabeled glucose. After the cultures have been grown for the targeted amount of time, the medium was aspirated completely, then cells were washed thrice with 5% D-mannitol solution, followed by inactivation with liquid nitrogen immediately. |
Sample Type: | Cultured cells |
Treatment:
Treatment ID: | TR001247 |
Treatment Summary: | One hour before the isotope switch, the medium was aspirated and replaced with fresh unlabeled medium[1]. Subsequently, VCaP and RWPE-1 were cultured in RPMI 1640 medium with 2 g/L [U−13C]-glucose and 10% dFBS for 0 h, 0.25 h, 4 h, 12 h, 24 h and long-term for 6 days performing thereby 3 passages. 10 cm petri dish with 10 mL of media was used for each sample, and 4 independent biological replicates were prepared for the labeling experiments. Unlabeled samples were treated in the same way with medium containing unlabeled glucose. |
Sample Preparation:
Sampleprep ID: | SP001240 |
Sampleprep Summary: | After the cultures have been grown for the targeted amount of time, the medium was aspirated completely, then cells were washed thrice with 5% D-mannitol solution, followed by inactivation with liquid nitrogen immediately. Cells were handled on ice during the preparation. 1 mL of precooled methanol, containing 13.3 μM of MetS and CSA, was added to each cell culture dish. Cells were harvested using a cell scraper, and transferred into a 5 mL tube. Then, 1 mL of chloroform was added and vortexed for 5 min, followed by 400 μL of Milli-Q water and 10 min of vortexing. After 5 min of standing, the mixture was centrifuged to form a two-phase system (10,000 g, 4 °C, 15 min). The upper layer was transferred and centrifugally filtered through a Millipore 5-kDa cutoff filter (USA) (13,000 g, 3h at 4°C). The filtrate was dried and stored at -80 °C. Finally, the upper extract was dissolved in Milli-Q water containing 50 μM of 3-AP, DPA, DHD and TMA for capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS) analysis. |
Combined analysis:
Analysis ID | AN001929 | AN001930 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | CE | CE |
Chromatography system | Agilent 1290 Infinity II | Agilent 1290 Infinity II |
Column | a fused silica capillary (50 μm i.d. × 80 cm) | a fused silica capillary (50 μm i.d. × 80 cm) |
MS Type | ESI | ESI |
MS instrument type | Triple quadrupole | Triple quadrupole |
MS instrument name | Agilent 6224 TOF | Agilent 6224 TOF |
Ion Mode | POSITIVE | NEGATIVE |
Units | Relative intensity after normalization | Relative intensity after normalization |
Chromatography:
Chromatography ID: | CH001400 |
Instrument Name: | Agilent 1290 Infinity II |
Column Name: | a fused silica capillary (50 μm i.d. × 80 cm) |
Column Temperature: | 20 |
Flow Rate: | a positive voltage of 27 kV |
Injection Temperature: | 20 |
Solvent A: | 1 M formic acid |
Chromatography Type: | CE |
Chromatography ID: | CH001401 |
Instrument Name: | Agilent 1290 Infinity II |
Column Name: | a fused silica capillary (50 μm i.d. × 80 cm) |
Column Temperature: | 20 |
Flow Rate: | internal pressure of 15 mbar and positive voltage of 30 kV |
Injection Temperature: | 20 |
Solvent A: | 100% water; 25 mM ammonium acetate; 75 mM ammonium phosphate, pH 8.5 |
Chromatography Type: | CE |
MS:
MS ID: | MS001785 |
Analysis ID: | AN001929 |
Instrument Name: | Agilent 6224 TOF |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | nebulizer pressure of 5 psig, dry gas temperature of 300 °C, nitrogen flow of 7 L/min, capillary voltage of 4 kV, fragmentor of 105 V, skimmer of 50 V, Oct RFV of 650 V, acquisition rate of 1.5 spectra/s and mass range of 60-1,000 were utilized. |
Ion Mode: | POSITIVE |
MS ID: | MS001786 |
Analysis ID: | AN001930 |
Instrument Name: | Agilent 6224 TOF |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | capillary voltage of 3.5 kV, fragmentor of 125 V and mass range of 50-1,000 were set in the TOF/MS analysis. During sample analysis, reference masses were applied to ensure the real-time calibration of exact mass measurement. |
Ion Mode: | NEGATIVE |