Return to study ST002697 main page
MB Sample ID: SA267124
| Local Sample ID: | C2-01-8889 |
| Subject ID: | SU002800 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | C57/Bl6 mice |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002800 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | C57/Bl6 mice |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| C2-01-8889 | SA267124 | FL034970 | Control | Group |
Collection:
| Collection ID: | CO002793 |
| Collection Summary: | Blood samples were taken on the day of sacrifice using the cardiac puncture procedure, which was conducted humanely under anesthesia, blood samples were then centrifuged at 2000 RCF for 10 minutes and the serum was transfered into a new tube and stored in -80 freezer. After animal sacrifice and tissue collection, tissues were snap frozen using liquid nitrogen and kept in a -80 freezer. |
| Sample Type: | Blood (serum) |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002809 |
| Treatment Summary: | Mice in the HU group were unloaded from their tails for three weeks. Mice in the HUR group were unloaded from their tails for three weeks before being reloaded into the ground for two weeks. |
Sample Preparation:
| Sampleprep ID: | SP002806 |
| Sampleprep Summary: | 100 µL from each plasma sample was transferred to Eppendorf, then a volume of 300 µL of methanol was added to the aliquot. Samples were vortexed, then chilled at -20 °C for 2 hours followed by centrifugation (14000 rpm, 15 minutes, 24°C) to precipitate protein. The metabolite-containing supernatants were collected and transferred to glass vials for drying using the EZ-2 Plus (GeneVac, Ipswich, UK) at 40 ±1°C and the protein pellets that remained were air dried for proteomics (see section 2.4). Dried metabolite samples were resuspended with 200 µl (0.1% formic acid in water), and vortexed for 2 min. The samples were filtered using a hydrophilic nylon syringe filter (0.45 μm pore size) and placed within glass inserts prior to being analyzed by Q-TOF MS. |
Combined analysis:
| Analysis ID | AN004371 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Bruker Elute |
| Column | Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Bruker timsTOF |
| Ion Mode | POSITIVE |
| Units | AU |
Chromatography:
| Chromatography ID: | CH003277 |
| Chromatography Summary: | Samples were chromatographically separated by inline reversed-phase chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm x 2.1 mm, 1.8µm beads) was maintained at 35C. For metabolomics, 10 µL was injected twice for each sample and eluted using a 30-minute gradient as follows: 1% ACN was held for 2 minutes, ramping to 99% ACN over 15 minutes, held at 99% ACN for 3 minutes before re-equilibrating to 1% ACN for 10 minutes. Flow rates were 250 µL/min for elution and 350 µL/min for re-equilibration. |
| Instrument Name: | Bruker Elute |
| Column Name: | Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 um) |
| Column Temperature: | 35 |
| Flow Gradient: | 1%B to 99%B in 15 min |
| Flow Rate: | 250 uL/min |
| Solvent A: | Water (0.1% Formic Acid) |
| Solvent B: | ACN (0.1% Formic Acid) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004119 |
| Analysis ID: | AN004371 |
| Instrument Name: | Bruker timsTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The MS analysis was performed using a TimsTOF (Bruker, Darmstadt, Germany) with Apollo II electrospray ionization (ESI) source. The drying gas was set to flow at 10 L/min and the drying temperature to 220C and the nebulizer pressure to 2.2 bar. The capillary voltage was 4500 V and the end plate offset 500V. For metabolomics the scan range was 20-1300 m/z. The collision energy was set to 20 eV, the cycle time to 0.5 seconds with a relative minimum intensity threshold of 400 counts per thousand and target intensity of 20,000. Sodium formate was injected as an external calibrant in the first 0.3 minutes of each LC-MS/MS run. MetaboScape 4.0 software was used for metabolite processing and statistical analysis (Bruker Daltonics). The following parameters for molecular feature identification and "bucketing" were set in the T-ReX 2D/3D workflow: For peak detection, a minimum intensity threshold of 1,000 counts is required, as well as a minimum peak duration of 7 spectra, with feature quantification determine using peak area. The file masses were recalibrated based on the external calibrant injected between 0-0.3 min. |
| Ion Mode: | POSITIVE |
