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MB Sample ID: SA353618
| Local Sample ID: | DUKE-17597 |
| Subject ID: | SU003373 |
| Subject Type: | Mammal |
| Subject Species: | Bos taurus |
| Taxonomy ID: | 9913 |
| Age Or Age Range: | 18-27 months |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003373 |
| Subject Type: | Mammal |
| Subject Species: | Bos taurus |
| Taxonomy ID: | 9913 |
| Age Or Age Range: | 18-27 months |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| DUKE-17597 | SA353618 | FL041246 | Bovine_Meat | Sample source |
| DUKE-17597 | SA353618 | FL041246 | Grain | Factor |
Collection:
| Collection ID: | CO003366 |
| Collection Summary: | All cattle were processed in USDA-inspected slaughter facilities and the researchers worked with the producers to collect meat samples (Pectoralis profundus) from 18 individual animals (n=18) per group for both the grass-fed and grain-fed group. Upon arrival at the lab, all meat samples were stored in a -40°C freezer and processed for analysis within 3 weeks of arrival. The meat samples were ground individually in a commercial meat grinder, and patties (112g) were cooked in a commercial oven (175 °C) until the internal temperature of the patties registered at 71 °C as determined by a meat thermometer. Thereafter ~2 grams from the center of each patty were obtained (n = 18 pasture-finished beef; n = 18 for grain-finished beef), immediately frozen in liquid nitrogen, and stored at -80 degrees °C until further analysis. |
| Sample Type: | Muscle |
| Collection Method: | Shipment on dry ice |
| Collection Location: | Columbus MT and Aberdeen, SD |
| Collection Frequency: | Once |
| Storage Conditions: | -80℃ |
| Collection Vials: | Vacuum-sealed bags |
| Storage Vials: | Cryovials |
Treatment:
| Treatment ID: | TR003382 |
| Treatment Summary: | All analyzed samples (pasture-finished and grain-finished) were from cattle harvested in September-October of 2020 and had a Black Angus genetic background. The pasture-finished animals were between 25-27 months of age, while the grain-finished animals ranged from 18-22 months of age at time of slaughter. The pasture-finished beef samples were sourced from Alderspring Ranch in May, Idaho, USA which employs adaptive grazing practices during rearing and finishing. During the spring/summer, the animals were rotationally grazed through 70 square miles of certified organic mountain rangeland in the Salmon Challis National Forest, ID where they had access to over 500 grasses, forbs, and shrub species from which to choose their diets. During the fall and winter, the cattle grazed organic home ranch pastures and/or ate certified organic hay at Alderspring Ranch, located at the Pahsimeroi Valley. The hay was harvested primarily from meadows at Alderspring Ranch and contained an estimated 20-40 different plant species, with dominant ones being dandelion grass (Taraxacum officinale), orchard grass (Dactylis glomerata), Kentucky bluegrass (Poa pratensis), sanfoin (Onobrychis viciifolia), alfalfa (Medicago sativa), and clover (Trifolium). The grain-finished beef samples were purchased from a distributor near Aberdeen, South Dakota, USA, who procure cattle from local feeder/finishing operations located within ~ 300 km from Aberdeen, SD. During the cow-calf and stocker phase, the cattle grazed on native pastures owned or leased by the feeder/finishing operations in Northern South Dakota, USA. The pastures contained an estimated 20-30 plant species, with dominant ones being big or sand bluestem (Andropogon spp), crested wheatgrass (Agropyron cristatum), annual brome (Bromus spp), blue grama (Bouteloua sp) and clover (Trifolium spp). During the finishing phase, the grain-finished cattle were kept in a feedlot located at the same feeding/finishing operation for ~ 130 days. |
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 |
Combined analysis:
| Analysis ID | AN005333 | AN005334 | AN005335 | AN005336 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase | HILIC |
| Chromatography system | Waters Acquity | Waters Acquity | Waters Acquity | Waters Acquity |
| Column | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) | Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) |
| MS Type | Other | Other | Other | Other |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | POSITIVE | NEGATIVE | NEGATIVE |
| Units | Arbitrary Units | Arbitrary Units | Arbitrary Units | Arbitrary Units |
Chromatography:
| Chromatography ID: | CH004035 |
| Chromatography Summary: | Low pH polar (LC/MS Pos early) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 5% B to 80% B over 3.35 minutes |
| Flow Rate: | 0.35 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Solvent B: | 100% methanol; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004036 |
| Chromatography Summary: | Low pH Lipophilic (LC/MS Pos late) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 40% B to 99.5% B over 1.0 minute, hold 99.5% B for 2.4 minutes. |
| Flow Rate: | 0.60 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Solvent B: | 50% methanol/50% acetonitrile; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004037 |
| Chromatography Summary: | High pH (LC/MS Neg) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 0.5 to 70% B over 4.0 minutes, then rapid gradient to 99% B in 0.5 minutes. |
| Flow Rate: | 0.35 mL/min |
| Solvent A: | 100% water; 6.5 mM ammonium bicarbonate, pH 8 |
| Solvent B: | 95% methanol/5% water; 6.5 mM ammonium bicarbonate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004038 |
| Chromatography Summary: | HILIC (LC/MS Polar Neg) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 5% B to 50% B in 3.5 minutes, then linear gradient from 50% B to 95% B in 2 minutes. |
| Flow Rate: | 0.50 mL/min |
| Solvent A: | 15% water/5% methanol/80% acetonitrile; 10 mM ammonium formate, (effective pH 10.16 with NH4OH) |
| Solvent B: | 50% water/50% acetonitrile; 10 mM ammonium formate, (effective pH 10.60 with NH4OH) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS005063 |
| Analysis ID: | AN005333 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Pos early) |
| Ion Mode: | POSITIVE |
| MS ID: | MS005064 |
| Analysis ID: | AN005334 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Pos late) |
| Ion Mode: | POSITIVE |
| MS ID: | MS005065 |
| Analysis ID: | AN005335 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Neg) |
| Ion Mode: | NEGATIVE |
| MS ID: | MS005066 |
| Analysis ID: | AN005336 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Polar) |
| Ion Mode: | NEGATIVE |
