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MB Sample ID: SA136219

Local Sample ID:P 161
Subject ID:SU001684
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:22 strains from the Collaborative Cross (CC) mouse panel

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Subject ID:SU001684
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:22 strains from the Collaborative Cross (CC) mouse panel


Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
P 161SA136219FL017482AIN-76ADiet
P 161SA136219FL017482baselineTimePoint


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 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. 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,3µm)
MS instrument type QTRAP
MS instrument name ABI Sciex API 4000 QTrap
Units micromolar


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,3µm)
Column Temperature:Room Temperature
Flow Rate:0.25 ml/min
Sample Injection:5 µL
Solvent A:0.1% acetic acid in water
Solvent B:0.1% acetic acid in methanol
Weak Wash Solvent Name:70% water, 20% methanol, 10% 2-propanol
Strong Wash Solvent Name:50:50 Acetonitrile:Methanol
Chromatography Type:Normal phase


MS ID:MS002452
Analysis ID:AN002640
Instrument Name:ABI Sciex API 4000 QTrap
Instrument Type:QTRAP
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.