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MB Sample ID: SA136317

Local Sample ID:56
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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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