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MB Sample ID: SA136363
Local Sample ID: | 5 |
Subject ID: | SU001684 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | 22 strains from the Collaborative Cross (CC) mouse panel |
Gender: | Female |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001684 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | 22 strains from the Collaborative Cross (CC) mouse panel |
Gender: | Female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
5 | SA136363 | FL017484 | H-Sucrose | Diet |
5 | SA136363 | FL017484 | postdiet | TimePoint |
Collection:
Collection ID: | CO001677 |
Collection Summary: | Blood was drawn from the mice after a 2-week acclimation period (baseline) and after 8 weeks (postdiet) on either a high protein (H-Protein) or high fat high sucrose (H-Sucrose). Blood samples were collected via retro-orbital bleed with heparinized capillary tubes into EDTA tubes, placed on ice, and centrifuged at 6000 rpm for 10 minutes at 4°C for plasma collection. Plasma was then transferred to 1.5 ml Eppendorf tubes and stored at -80°C. |
Sample Type: | Blood (plasma) |
Collection Method: | retro-orbital bleed with heparinized capillary tubes |
Storage Conditions: | Described in summary |
Collection Vials: | EDTA tubes |
Treatment:
Treatment ID: | TR001697 |
Treatment Summary: | Approximately 4-5 mice were assigned to either the high protein (H-Protein) or high fat high sucrose (H-Sucrose) diet per strain for 8 weeks. |
Treatment: | Diet challenge |
Sample Preparation:
Sampleprep ID: | SP001690 |
Sampleprep Summary: | Baseline and post-diet circulating trimethylamine N-oxide (TMAO), choline, phosphocholine, betaine, and carnitine were quantified using liquid chromatography–mass spectrometry (LC-MS) methods described by Wang et al. (2014) with modifications (Wang et al., 2014. Analytical Biochemistry 455 (June 2014): 35–40. https://doi.org/10.1016/j.ab.2014.03.016.). Briefly, samples (20 µl plasma) were aliquoted to a 2 ml Eppendorf tube and mixed with 80 µl of 5 µM surrogate standard comprised of deuterated analytes in methanol. Standards ranging from 0 µM to 100 µM of non-deuterated analytes in methanol were run in order to establish analyte standard curves. Two-fold serial dilutions of a 100 µM stock solution in methanol was used to make 13 standards. To prepare standards for sample quantification, 80 µl of 5 µM SSTD and 20 µl of each standard were aliquoted directly to the glass inserts in HPLC vials and briefly vortexed. Prior to acquisition, samples and standards were vortexed for 30 seconds and centrifuged at 18,000 g at 10°C for 10 min. Supernatant (5 µl) was transferred to 150 µl glass inserts in High Performance Liquid Chromatography (HPLC) vials and analyzed by injection onto a silica column (150 by 2 mm, 3 um particle Silica (2) with 100 Angstrom; Catalog #00F-41620-B0, Phenomenex, Torrance, CA) at a flow rate of 0.25 ml/min using a Waters Acquity UPLC (Waters, Milford, MA) interfaced with an API 4000 Q-TRAP mass spectrometer (AB SCIEX, Framingham, MA). A discontinuous gradient was generated to resolve the analytes by mixing solvent A (0.1% acetic acid in water) with solvent B (0.1% acetic acid in methanol) at different ratios starting from 2% B linearly to 15% B over 5 min, then linearly to 100% B to 6.25 min, then hold to 8 min, and then back to 2% B at 6.25 min and held until 10 min. |
Sampleprep Protocol Filename: | phoebeyam_SP_protocol.pdf |
Combined analysis:
Analysis ID | AN002640 |
---|---|
Analysis type | MS |
Chromatography type | Normal phase |
Chromatography system | Waters Acquity UPLC |
Column | Luna Silica (150 x 2mm,3m) |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | ABI Sciex API 4000 QTrap |
Ion Mode | POSITIVE |
Units | micromolar |
Chromatography:
Chromatography ID: | CH001950 |
Chromatography Comments: | Phenomenex Luna 3 µm Silica (2) 100 Å, LC Column 150 x 2 mm |
Instrument Name: | Waters Acquity UPLC |
Column Name: | Luna Silica (150 x 2mm,3m) |
Column Temperature: | Room Temperature |
Flow Rate: | 0.25 ml/min |
Sample Injection: | 5 µL |
Solvent A: | 100% water; 0.1% acetic acid |
Solvent B: | 100% methanol; 0.1% acetic acid |
Weak Wash Solvent Name: | 70% water, 20% methanol, 10% 2-propanol |
Strong Wash Solvent Name: | 50:50 Acetonitrile:Methanol |
Chromatography Type: | Normal phase |
MS:
MS ID: | MS002452 |
Analysis ID: | AN002640 |
Instrument Name: | ABI Sciex API 4000 QTrap |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | Analytes were monitored using electrospray ionization in positive-ion mode with multiple reaction monitoring (MRM) of precursor and characteristic production transitions as shown in MS_protocol.pdf. The parameters for the ion monitoring were as follows: spray voltage, 4.5 kV; curtain gas, 15; GS1, 60; GS2, 50; CAD gas, medium; Nitrogen (99.95% purity) was used as the source and collision gas. Integration and quantification of values was done using Analyst 1.6.2 software (AB SCIEX, Singapore). Standard linearity was calculated using linear regression model. Please see LC_protocol.pdf and MS_protocol.pdf for additional details. |
Ion Mode: | POSITIVE |
Analysis Protocol File: | phoebeyam_LC_protocol.pdf |