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MB Sample ID: SA353626
| Local Sample ID: | DUKE-17572 |
| Subject ID: | SU003373 |
| Subject Type: | Mammal |
| Subject Species: | Bos taurus |
| Taxonomy ID: | 9913 |
| Age Or Age Range: | 18-27 months |
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Sample Preparation:
| Sampleprep ID: | SP003380 |
| Sampleprep Summary: | Samples analyzed for untargeted metabolomic profiling through collaborations with Metabolon (Morrisville, NC). One hundred (100 mg) was weighed out for each sample and recovery standards were added for quality control purposes. Proteins were subsequently precipitated with methanol under vigorous shaking for 2 min (Glen Mills Geno Grinder 2000, Clifton, NJ, USA) followed by centrifugation (15,000 × g). The resulting extract was divided into five fractions: two for analysis by separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample for backup. Sample extracts were placed briefly on a TurboVap (Zymark) to remove the organic solvent and reconstituted in mobile phases described below. The UPLC-MS/MS platform utilized a Waters Acquity UPLC with Waters UPLC BEH C18-2.1×100 mm, 1.7 μm columns and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer. One aliquot was analyzed using acidic positive ion conditions, which was chromatographically optimized for more hydrophilic compounds. The extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). The second aliquot was also analyzed using acidic positive ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. The extract was gradient eluted from the same C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA. The third aliquot was analyzed using basic negative ESI-optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water with 6.5 mmol/L Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ESI following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10 mmol/L Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion, while the scan range covered m/z 70–1000 at a resolving power of R=35,000 optimized at fifty percent of the maximum peak height (FWHM). Metabolites were identified by automated comparison of the ion features in the samples to a reference library of chemical standard entries that considered the retention time, molecular weight (m/z), preferred adducts, in-source fragments, and associated MS spectra77. The data were curated by visual inspection for quality control using Metabolon’s proprietary software. Library matches for each compound were checked for each sample and corrected if necessary. Peaks were quantified using area-under-the-curve. A data normalization step was performed to correct for variation resulting from instrument inter-day tuning differences by setting the medians to equal one (1.00) and normalizing each data point proportionately (termed “block correction”). This preserved variation between samples while allowing metabolites of different raw peak areas to be compared on a similar graphical scale. |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Methanol and methanol/water/dichloromethane |
| Extract Storage: | On ice |
| Sample Spiking: | Deuterated standards |
