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MB Sample ID: SA214158
| Local Sample ID: | AP02_20 |
| Subject ID: | SU002328 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | Mammals |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002328 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | Mammals |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| AP02_20 | SA214158 | FL026021 | normoxia | Treatment |
| AP02_20 | SA214158 | FL026021 | mitochondrial | Cellular fraction |
Collection:
| Collection ID: | CO002321 |
| Collection Summary: | To isolate mitochondrial and cytosolic cellular fractions we followed a previously published rapid subcellular fractionation method (Lee et al., 2019). Briefly, on the day of the experiment, cells were washed twice with ice-cold PBS and scraped from the flasks with 2 ml PBS. 1:4 of cells was transferred to a tube and served as the whole cell lysate (WCL). After a brief centrifugation step (10,000 g, 3 minutes, 4 °C), supernatant was removed and 300 ul of metabolite extraction solution (50%MetOH:30%acetonitrile:20% ultrapure water) was added to samples. Samples were incubated with the extraction buffer for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction. The rest 3:4 of cells was subjected to digitonin-based cellular fractionation. More precisely, after a brief centrifugation step (10,000 g, 10 sec, 4 °C), supernatant was removed and the pellet was resuspended in 1 ml of ice-cold digitonin-PBS buffer (1mg/ml). Following quick centrifugation (10,000 g, 10 sec, 4 °C), supernatant and pellet were collected as the cytosolic and mitochondrial fractions, respectively. 4 ml of 50%MetOH:30%acetonitrile was added in the cytosolic fractions to extract metabolites. Samples were incubated for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction. The mitochondrial fraction was resuspended in 100 ul metabolite extraction solution and incubated for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR002340 |
| Treatment Summary: | Commercially available MSCs were cultured in T175 flasks under normoxic or hypoxic conditions for seven days, in cell culture medium (MEM-Alpha supplemented with 10% FBS and 1% penicillin/streptomycin). |
Sample Preparation:
| Sampleprep ID: | SP002334 |
| Sampleprep Summary: | The cell extract suspension was incubated for 15 minutes, at 4 °C, shaking in a thermomixer at maximum speed. Samples were then centrifugated for 20 minutes at 4 °C, at 13,000 rpm. The top 80% of the supernatant was collected and subjected to LC/MS analysis. |
Combined analysis:
| Analysis ID | AN003660 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | SeQuant ZIC-pHILIC |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Orbitrap Exploris 240 |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002712 |
| 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 |
| 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: | MS003411 |
| Analysis ID: | AN003660 |
| Instrument Name: | Thermo Orbitrap 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. 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. Mild trapping was enabled. Metabolite identification was performed in the Compound Discoverer software (v 3.2, Thermo Fisher Scientific). Metabolite identities were confirmed using the following parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) fragment ions were matched within 5 pm to an in-house spectral library of authentic compound standards analysed with the same ddMS2 method with a best match score of over 70; (3) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. Chromatogram review and peak area integration were performed using the Tracefinder software (v 5.0, Thermo Fisher Scientific) and the peak area for each detected metabolite was normalized against the total ion count (TIC) of that sample to correct any variations introduced from sample handling to instrument analysis. The normalized areas were used as variables for further statistical data analysis. |
| Ion Mode: | UNSPECIFIED |
