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MB Sample ID: SA300806
| Local Sample ID: | G31-Con-31A3B |
| Subject ID: | SU002909 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Age Or Age Range: | 6 months |
| Weight Or Weight Range: | 27-43 |
| Gender: | Male |
| Animal Animal Supplier: | The Jackson Laboratory |
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Subject:
| Subject ID: | SU002909 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Age Or Age Range: | 6 months |
| Weight Or Weight Range: | 27-43 |
| Gender: | Male |
| Animal Animal Supplier: | The Jackson Laboratory |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| G31-Con-31A3B | SA300806 | FL036536 | Wild-type | Genotype |
Collection:
| Collection ID: | CO002902 |
| Collection Summary: | Serum samples from each mouse were collected from heart blood immediately after carbon dioxide euthanasia. |
| Sample Type: | Blood (serum) |
Treatment:
| Treatment ID: | TR002918 |
| Treatment Summary: | No treatment. |
Sample Preparation:
| Sampleprep ID: | SP002915 |
| Sampleprep Summary: | Serum samples from each mouse were thawed and 20 μL was consumed to extract metabolites for each mice. Metabolite extraction was facilitated by adding 180 μL methanol containing stable isotope-labeled chemicals ([D5]-glutamine, [D2]-𝛾-aminobutyric acid, [D3]-tryptophan, [D2]-indole-3-propionic acid, [D2]-indole-3-acetic acid, [D13]-acetylcholine in 500 nM; [D4]-serotonin in 250 nM; and [D4]-kynurenic acid in 50 nM), and incubated under -20°C for 1 hr, followed by centrifugation under 15,000 ×g, 4°C for 10 minutes to collect the supernatant (150 μL). The supernatant was dried by SpeedVac® and reconstituted with 100 μL 2% acetonitrile in water. |
Combined analysis:
| Analysis ID | AN004558 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | m/z |
Chromatography:
| Chromatography ID: | CH003425 |
| Chromatography Summary: | The serum analytes were injected (10 μL) into a Waters Acquity UPLC HSS T3 (reverse phase C18, 100 Å, 1.8 μm, 2.1 mm × 100 mm) analytical column controlled at 40 °C, with the mobile phase composed of water (A) and acetonitrile (B) both added with 0.1% formic acid at a flow rate of 0.4 mL/min. The 15-min-gradient for chromatographic separation was set as the following: 2% B from 0-1 min; 2%-15% B from 1-3 min; 15%-50% B from 3-6 min; 50%-98% B from 6-7.5 min; 98% B held from 7.5-11.5 min; 98%-2% B from 11.5-11.6 min; and 2% B held from 11.6-15 min for a final re-equilibration. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
| Column Temperature: | 40 |
| Flow Gradient: | 2% B from 0-1 min; 2%-15% B from 1-3 min; 15%-50% B from 3-6 min; 50%-98% B from 6-7.5 min; 98% B held from 7.5-11.5 min; 98%-2% B from 11.5-11.6 min; and 2% B held from 11.6-15 min for a final re-equilibration. |
| Flow Rate: | 0.4 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004305 |
| Analysis ID: | AN004558 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The mass spectrometry was set to scan under the positive mode with the sheath gas, auxiliary gas, and sweep gas set to flow rates of 50, 13, and 3 psi, respectively. With the spray voltage set to 3.5 kV, the capillary and auxiliary gas heating temperature were respectively controlled to 263°C and 425°C to fully scan across m/z 70 to 1,000. The resolution was set to 70,000 FWHM (m/z 200). The automatic gain control (AGC) and the maximal injection time (MIT) was set to 2×105 and 50 msec, respectively. Routine mass calibrations were conducted before and after the sample analysis. The samples were blocked-randomized for the injection order. Quality control samples for the serum analytes were prepared by pooling the aliquots of each sample. Method blank samples were prepared for the serum samples by surrogating the biospecimen with water and following the same experimental procedures. If MS/MS spectrums were to be collected, the parallel reaction monitoring (PRM) mode was used, with the isolation width, AGC, and MIT set to 1.2, 3×105 and 100 msec, respectively, at the resolution of 17,500 FWHM (m/z 200). |
| Ion Mode: | POSITIVE |
