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MB Sample ID: SA210721
| Local Sample ID: | CR02_047 |
| Subject ID: | SU002285 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002285 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CR02_047 | SA210721 | FL025740 | Fh1-/-CL1 | Genotype |
| CR02_047 | SA210721 | FL025740 | siFoxa2 | Treatment |
Collection:
| Collection ID: | CO002278 |
| Collection Summary: | 5 x 104 Fh1fl/fl and 1 x 105 Fh1-/-CL1 and Fh1-/-CL19 cells were plated the onto 6-well plate and reverse transfected with siRNA. Before extraction, cells were counted using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept on cold bath with dry ice and methanol. Metabolite extraction buffer (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well following the proportion 1×106 cells/0.5 ml of buffer. Plates were kept at −80°C and kept overnight. The following day, the extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 10 min at 4°C, the supernatants were transferred into LC-MS vials. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR002297 |
| Treatment Summary: | 5 x 104 Fh1fl/fl and 1 x 105 Fh1-/-CL1 and Fh1-/-CL19 cells were reverse transfected in 6-well plates with 50 pmol of either control siNT (Horizon, D-001810-10-05) or siFoxa2 (Horizon, L-043601-00-0005) siRNA using 2.5 µl RNAiMAX per well. Cells were incubated for 72 hours before either RNA or protein were extracted. |
Sample Preparation:
| Sampleprep ID: | SP002291 |
| Sampleprep Summary: | 5 x 104 Fh1fl/fl and 1 x 105 Fh1-/-CL1 and Fh1-/-CL19 cells were plated the onto 6-well plate and reverse transfected with siRNA. Before extraction, cells were counted using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept on cold bath with dry ice and methanol. Metabolite extraction buffer (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well following the proportion 1×106 cells/0.5 ml of buffer. Plates were kept at −80°C and kept overnight. The following day, the extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 10 min at 4°C, the supernatants were transferred into LC-MS vials. |
Combined analysis:
| Analysis ID | AN003599 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish Horizon |
| Column | SeQuant ZIC-pHILIC |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Exploris 240 |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002659 |
| Chromatography Summary: | Chromatographic separation of metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B. Samples were randomized and the injection volume was 5 µl. A pooled quality control (QC) sample was generated from an equal mixture of all individual samples and analysed interspersed at regular intervals. |
| Instrument Name: | Thermo Vanquish Horizon |
| Column Name: | SeQuant ZIC-pHILIC |
| Column Temperature: | 40 |
| Flow Gradient: | 0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B |
| Flow Rate: | 0.200 mL/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003354 |
| Analysis ID: | AN003599 |
| Instrument Name: | Thermo Exploris 240 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Metabolites were measured with Vanquish Horizon UHPLC coupled to an Orbitrap Exploris 240 mass spectrometer (both Thermo Fisher Scientific) via a heated electrospray ionization source. The spray voltages were set to +3.5kV/-2.8 kV, RF lens value at 70, the heated capillary held at 320 °C, and the auxiliary gas heater held at 280 °C. The flow rate for sheath gas, aux gas and sweep gas were set to 40, 15 and 0, respectively. For MS1 scans, mass range was set to m/z=70-900, AGC target set to standard and maximum injection time (IT) set to auto. Data acquisition for experimental samples used full scan mode with polarity switching at an Orbitrap resolution of 120000. Data acquisition for untargeted metabolite identification was performed using the AcquireX Deep Scan workflow, an iterative data-dependent acquisition (DDA) strategy using multiple injections of the pooled sample. In brief, sample was first injected in full scan-only mode in single polarity to create an automated inclusion list. MS2 acquisition was then carried out in triplicate, where ions on the inclusion list were prioritized for fragmentation in each run, after which both the exclusion and inclusion lists were updated in a manner where fragmented ions from the inclusion list were moved to exclusion list for the next run. DDA full scan-ddMS2 method for AcquireX workflow used the following parameters: full scan resolution was set to 60000, fragmentation resolution to 30000, fragmentation intensity threshold to 5.0e3. Dynamic exclusion was enabled after 1 time and exclusion duration was 10s. Mass tolerance was set to 5ppm. Isolation window was set to 1.2 m/z. Normalized HCD collision energies were set to stepped mode with values at 30, 50, 150. Fragmentation scan range was set to auto, AGC target at standard and max IT at auto. Xcalibur AcquireX method modification was on. Mild trapping was enabled. Metabolite identification was performed in the Compound Discoverer software (v 3.2, Thermo Fisher Scientific). Metabolites were annotated at the MS2 level using both an in-house mzVault spectral database curated from 1051 authentic compound standards and the online spectral library mzCloud. The precursor mass tolerance was set to 5 ppm and fragment mass tolerance set to 10 ppm. Only metabolites with mzVault or mzCloud best match score above 50% and 75%, respectively, and RT tolerance within 0.5 min to that of a purified standard run with the same chromatographic method were exported to generate a list including compound names, molecular formula and RT. The curated list was then used for further processing in the Tracefinder software (v 5.0, Thermo Fisher Scientific), where extracted ion chromatographs for all compound were examined and manually integrated if necessary. False positive, noise or chromatographically unresolved compounds were removed. The peak area for each detected metabolite was then normalized against the total ion count (TIC) of that sample to correct any variations introduced from sample handling through instrument analysis. The normalized areas were used as variables for further statistical data analysis. |
| Ion Mode: | UNSPECIFIED |
