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MB Sample ID: SA300804

Local Sample ID:G31-Con-31A2A
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 IDMB Sample IDFactor Level IDLevel ValueFactor Name
G31-Con-31A2ASA300804FL036536Wild-typeGenotype

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
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