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MB Sample ID: SA217835
| Local Sample ID: | SPT_WT_15min_0131111 |
| Subject ID: | SU002360 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC, CCL-247 |
| Cell Strain Details: | HCT116 |
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Subject:
| Subject ID: | SU002360 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC, CCL-247 |
| Cell Strain Details: | HCT116 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| SPT_WT_15min_0131111 | SA217835 | FL026448 | Wild-type | Genotype |
| SPT_WT_15min_0131111 | SA217835 | FL026448 | 15min | Treatment |
Collection:
| Collection ID: | CO002353 |
| Collection Summary: | For tracing studies with [U-13C16]palmitate, HCT116 cells were cultured in growth medium in the presence of 0.1 μg/ml doxycycline for 7 days before tracer start. Growth medium was replaced to DMEM medium containing 1% (v/v) delipidated FBS 24h prior tracer start and medium exchange again 1h prior tracer trace. [U-13C16]palmitate was noncovalently bound to fatty acid-free BSA and added to culture medium at 5% of the final volume (50 μM final concentration). Media was prewarmed to 37oC in a cell incubator with 5% CO2 and cells were traced for 15 min, 1 h, and 4 h. For targeted sphingolipid analysis, cells were washed with 0.9% (w/v) NaCl and extracted with 0.25 mL of −20°C methanol, 0.25 mL -20°C chloroform, and 0.1 mL of water. The tubes were vortexed for 5 min, centrifuged at 20,000 x g at 4°C for 5 min, and the lower organic phase was collected. The remaining polar phase was extracted with 2 μL formic acid and 0.25 mL of -20 °C chloroform. The organic phases were combined, dried under air, resuspended in 80 μL Buffer B (0.2% formic acid and 1 mM ammonium formate in methanol), sonicated for 10 min, and centrifuged for 10 min at 20,000 x g at 4 °C. The supernatant was transferred to LC vials containing glass inserts for analysis on a Q-Exactive LC-MS system. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR002372 |
| Treatment Summary: | HCT116 expressing SPTLC1WT or SPTLC1C133W were cultured in media containing [U-13C16]palmitate for 15min, 1h, and 4h. |
Sample Preparation:
| Sampleprep ID: | SP002366 |
| Sampleprep Summary: | The samples were reconstituted in 80 μL Buffer B (0.2% formic acid and 1 mM ammonium formate in methanol), sonicated for 10 min, and centrifuged for 10 min at 20,000 x g at 4 °C. The supernatant was transferred to LC vials containing glass inserts for analysis on a Q-Exactive LC-MS system. |
Combined analysis:
| Analysis ID | AN003716 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Kinetex C8 (150 x 3mm,2.6um,100Å) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | ion counts |
Chromatography:
| Chromatography ID: | CH002753 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Kinetex C8 (150 x 3mm,2.6um,100Å) |
| Column Temperature: | 40 |
| Flow Gradient: | The LC gradient held at 82% B for 0-3 min, then ran from 82%-90% B in 3-4 min, then 90-99% in 4-18 min, held at 99% B for 7 min, then reduced from 99%-82% from 25-27 min, then held at 82% for a further 13 mins. |
| Flow Rate: | 0.5 mL/min |
| Solvent A: | 100% water; 0.2% formic acid; 2 mM ammonium formate |
| Solvent B: | 100% methanol; 0.2% formic acid; 1 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003465 |
| Analysis ID: | AN003716 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | To quantify labeling on SL and deoxySL species from [U-13C16]palmitate a Q Exactive orbitrap mass spectrometer with a Vanquish Flex Binary UHPLC system (Thermo Scientific) was used with a Kinetex 2.6 μM C8 100 Å 150 x 3 mm LC column (Phenomenex) at 40°C. 5 μL of sample was injected. Chromatography was performed using a gradient of 2 mM ammonium formate and 0.2 % formic acid (mobile phase A) and 1 mM ammonium formate and 0.2 % formic acid in methanol (mobile phase B), at a flow rate of 0.5 mL/min. The LC gradient held at 82% B for 0-3 min, then ran from 82%-90% B in 3-4 min, then 90-99% in 4-18 min, held at 99% B for 7 min, then reduced from 99%-82% from 25-27 min, then held at 82% for a further 13 mins. Lipids were analyzed in positive mode using spray voltage 3 kV. Sweep gas flow was 5 arbitrary units, auxiliary gas flow 7 arbitrary units and sheath gas flow 50 arbitrary units, with a capillary temperature of 300°C. Full MS (scan range 150-2000 m/z) was used at 70 000 resolution with 1e6 automatic gain control and a maximum injection time of 200 ms. Data dependent MS2 (Top 6) mode at 17 500 resolution with automatic gain control set at 1e5 with a maximum injection time of 50 ms was used for peak identification, combined with known standards where possible. |
| Ion Mode: | POSITIVE |
