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MB Sample ID: SA174339
| Local Sample ID: | KG_3 |
| Subject ID: | SU001937 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | DSMZ |
| Cell Strain Details: | 8988T |
| Subject Comments: | pancreatic ductal adenocarcinoma |
| Cell Counts: | 1-2x10^6 |
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Subject:
| Subject ID: | SU001937 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | DSMZ |
| Cell Strain Details: | 8988T |
| Subject Comments: | pancreatic ductal adenocarcinoma |
| Cell Counts: | 1-2x10^6 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| KG_3 | SA174339 | FL021391 | KG (1 mM) | Treatment |
Collection:
| Collection ID: | CO001930 |
| Collection Summary: | Metabolites were initially extracted from samples by quickly aspirating the cell culture media and adding 1 mL of extraction buffer, consisting of 80% methanol (Fisher Scientific) and 500 nM metabolomics amino acid mix standard (Cambridge Isotope Laboratories). To effectively scale all harvested samples to equivalent volumes of extraction buffer, samples were fully dried down by Speedvac (Thermo Fisher, Waltham, MA) and reconstituted volumetrically by mixing the entire dried cell pellet sample with 1 mL of 80% methanol without QC standards in 2.0 mL screw cap vials containing ~100 µL of disruption beads (Research Products International, Mount Prospect, IL). Samples were scaled to a ratio of 1e6 cells to 1 mL of extraction solvent with all steps being carried out in a cold room. Each was homogenized for 10 cycles on a bead blaster homogenizer (Benchmark Scientific, Edison, NJ). Cycling consisted of a 30 sec homogenization time at 6 m/s followed by a 30 sec pause. Samples were subsequently spun at 21,000 x g for 3 min at 4°C. A set volume of each (450 µL) was transferred to a 1.5 mL tube and dried down by Speedvac concentration. Samples were reconstituted in 50 µL of Optima LC/MS grade water (Fisher Scientific, Waltham, MA). Samples were sonicated for 2 mins, then centrifuged at 21,000 x g for 3 min at 4°C. Twenty microliters were transferred to LC vials containing glass inserts for analysis. The remaining sample was placed in -80°C for long term storage. |
| Sample Type: | Cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001949 |
| Treatment Summary: | 8988T cells were treated with methyl acetate, which is used as a vehicle, 1 mM of alpha-ketoglutarate disodium salt, or 1 mM of dimethyl-alpha-ketoglutarate (prepared in methyl acetate) for 3 hours in DMEM supplemented with 10% dialyzed fetal bovine serum and 5% penstrep |
Sample Preparation:
| Sampleprep ID: | SP001943 |
| Sampleprep Summary: | Dried samples were reconstituted in 50 µL of Optima LC/MS grade water (Fisher Scientific, Waltham, MA). Samples were sonicated for 2 mins, then centrifuged at 21,000 x g for 3 min at 4°C. Twenty microliters were transferred to LC vials containing glass inserts for analysis. The remaining sample was placed in -80°C for long term storage. |
Combined analysis:
| Analysis ID | AN003015 | AN003016 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | ion counts | ion counts |
Chromatography:
| Chromatography ID: | CH002234 |
| Chromatography Summary: | Samples were subjected to an LC-MS analysis to detect and quantify known peaks. A metabolite extraction was carried out on each sample by quickly aspirating experimental media and adding 1 mL of 80% methanol containing internal QC standards. The LC column was a MilliporeTM ZIC-pHILIC (2.1 x150 mm, 5 μm) coupled to a Dionex Ultimate 3000TM system and the column oven temperature was set to 25oC for the gradient elution. A flow rate of 100 μL/min was used with the following buffers; A) 10 mM ammonium carbonate in water, pH 9.0, and B) neat acetonitrile. The gradient profile was as follows; 80-20%B (0-30 min), 20-80%B (30-31 min), 80-80%B (31-42 min). Injection volume was set to 2 μL for all analyses (42 min total run time per injection). |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 25 |
| Flow Rate: | 100 uL/min |
| Solvent A: | 100% water; 10 mM ammonium carbonate, pH 9.0 |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002804 |
| Analysis ID: | AN003015 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS analyses were carried out by coupling the LC system to a Thermo Q Exactive HFTM mass spectrometer operating in heated electrospray ionization mode (HESI). Method duration was 30 min with a polarity switching data-dependent Top 5 method for both positive and negative modes. Spray voltage for both positive and negative modes was 3.5 kV and capillary temperature was set to 320oC with a sheath gas rate of 35, aux gas of 10, and max spray current of 100 μA. The full MS scan for both polarities utilized 120,000 resolution with an AGC target of 3e6 and a maximum IT of 100 ms, and the scan range was from 67-1000 m/z. Tandem MS spectra for both positive and negative mode used a resolution of 15,000, AGC target of 1e5, maximum IT of 50 ms, isolation window of 0.4 m/z, isolation offset of 0.1 m/z, fixed first mass of 50 m/z, and 3-way multiplexed normalized collision energies (nCE) of 10, 30, 80. The minimum AGC target was 1e4 with an intensity threshold of 2e5. All data were acquired in profile mode. The resulting ThermoTM RAW files were read with ThermoFisher CommonCore RawFileReader, and an in-house python script (Skeleton) was used for peak detection and quantification of all internal standards and sample peaks based on a previously established library of metabolite retention times and accurate masses adapted from the Whitehead Institute, and verified with authentic standards and/or high resolution MS/MS spectral manually curated against the NIST14MS/MS and METLIN (2017) tandem mass spectral libraries. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS002805 |
| Analysis ID: | AN003016 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS analyses were carried out by coupling the LC system to a Thermo Q Exactive HFTM mass spectrometer operating in heated electrospray ionization mode (HESI). Method duration was 30 min with a polarity switching data-dependent Top 5 method for both positive and negative modes. Spray voltage for both positive and negative modes was 3.5 kV and capillary temperature was set to 320oC with a sheath gas rate of 35, aux gas of 10, and max spray current of 100 μA. The full MS scan for both polarities utilized 120,000 resolution with an AGC target of 3e6 and a maximum IT of 100 ms, and the scan range was from 67-1000 m/z. Tandem MS spectra for both positive and negative mode used a resolution of 15,000, AGC target of 1e5, maximum IT of 50 ms, isolation window of 0.4 m/z, isolation offset of 0.1 m/z, fixed first mass of 50 m/z, and 3-way multiplexed normalized collision energies (nCE) of 10, 30, 80. The minimum AGC target was 1e4 with an intensity threshold of 2e5. All data were acquired in profile mode. The resulting ThermoTM RAW files were read with ThermoFisher CommonCore RawFileReader, and an in-house python script (Skeleton) was used for peak detection and quantification of all internal standards and sample peaks based on a previously established library of metabolite retention times and accurate masses adapted from the Whitehead Institute, and verified with authentic standards and/or high resolution MS/MS spectral manually curated against the NIST14MS/MS and METLIN (2017) tandem mass spectral libraries. |
| Ion Mode: | POSITIVE |
