Summary of Study ST003254

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002020. The data can be accessed directly via it's Project DOI: 10.21228/M8HC1Q This work is supported by NIH grant, U2C- DK119886.

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Study IDST003254
Study TitleThe impact of grass- and grain-finishing on metabolomic profiles of North American Black Angus Beef cattle.
Study SummaryThe goal of this study was to compare meat metabolomes (pectoralis profundus) of Black Angus cattle from two commercial US beef finishing systems (pasture-finished on Western U.S. rangeland; n=18 and grain-finished in a Midwest U.S. feedlot; n=18).
Institute
Duke University
DepartmentSchool of Medicine
LaboratoryDuke Molecular Physiology Institute
Last Namevan Vliet
First NameStephan
Address300 N Duke St, Durham, NC 27701
Emailstephan.vanvliet@usu.edu
Phone2177785001
Submit Date2024-06-05
Num Groups2
Total Subjects36
Analysis Type DetailOther
Release Date2024-06-18
Release Version1
Stephan van Vliet Stephan van Vliet
https://dx.doi.org/10.21228/M8HC1Q
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002020
Project DOI:doi: 10.21228/M8HC1Q
Project Title:Analysis of Pasture-Fed vs Grain-Fed Meat in North American Ruminants
Project Summary:The goal of this work was to compare meat metabolomes (pectoralis profundus) of Black Angus cattle from two commercial US beef finishing systems (pasture-finished on Western U.S. rangeland; n=18 and grain-finished in a Midwest U.S. feedlot; n=18). 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. 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. Metabolomics analysis was performed by Metabolon (Morrisville, NC). Statistical analyses were performed in ArrayStudio/Jupyter Notebook, MetaboAnalyst, and R.
Institute:Duke University
Department:School of Medicine
Laboratory:Duke Molecular Physiology Institute
Last Name:van Vliet
First Name:Stephan
Address:300 N Duke St, Durham, NC 27701
Email:stephan.vanvliet@usu.edu
Phone:2177785001
Funding Source:USDA [grant no. 58-3064-0-003;2021-67034-35118] and the Dixon Water Foundation

Subject:

Subject ID:SU003373
Subject Type:Mammal
Subject Species:Bos taurus
Taxonomy ID:9913
Age Or Age Range:18-27 months

Factors:

Subject type: Mammal; Subject species: Bos taurus (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Factor
SA353613DUKE-17584Bovine_Meat Grain
SA353614DUKE-17605Bovine_Meat Grain
SA353615DUKE-17603Bovine_Meat Grain
SA353616DUKE-17601Bovine_Meat Grain
SA353617DUKE-17599Bovine_Meat Grain
SA353618DUKE-17597Bovine_Meat Grain
SA353619DUKE-17595Bovine_Meat Grain
SA353620DUKE-17593Bovine_Meat Grain
SA353621DUKE-17591Bovine_Meat Grain
SA353622DUKE-17587Bovine_Meat Grain
SA353623DUKE-17588Bovine_Meat Grain
SA353624DUKE-17582Bovine_Meat Grain
SA353625DUKE-17581Bovine_Meat Grain
SA353626DUKE-17572Bovine_Meat Grain
SA353627DUKE-17573Bovine_Meat Grain
SA353628DUKE-17579Bovine_Meat Grain
SA353629DUKE-17575Bovine_Meat Grain
SA353630DUKE-17576Bovine_Meat Grain
SA353631DUKE-17596Bovine_Meat Grass
SA353632DUKE-17604Bovine_Meat Grass
SA353633DUKE-17602Bovine_Meat Grass
SA353634DUKE-17574Bovine_Meat Grass
SA353635DUKE-17600Bovine_Meat Grass
SA353636DUKE-17598Bovine_Meat Grass
SA353637DUKE-17594Bovine_Meat Grass
SA353638DUKE-17577Bovine_Meat Grass
SA353639DUKE-17583Bovine_Meat Grass
SA353640DUKE-17578Bovine_Meat Grass
SA353641DUKE-17592Bovine_Meat Grass
SA353642DUKE-17590Bovine_Meat Grass
SA353643DUKE-17589Bovine_Meat Grass
SA353644DUKE-17571Bovine_Meat Grass
SA353645DUKE-17580Bovine_Meat Grass
SA353646DUKE-17586Bovine_Meat Grass
SA353647DUKE-17585Bovine_Meat Grass
SA353648DUKE-17570Bovine_Meat Grass
Showing results 1 to 36 of 36

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 ESI ESI ESI ESI
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:ESI
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:ESI
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:ESI
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:ESI
MS Comments:Metabolon (LC/MS Polar)
Ion Mode:NEGATIVE
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