Sample Collection
Samples used for this study were obtained as part of the HoloFish project (Norwegian Seafood Research Fund, project no. 901436). This cohort has been described previously 19. Briefly, we sampled 460 ready-to-harvest Atlantic salmon from a commercial production site close to Bergen, Norway, owned by Leroy Seafood Group in April 2018. Samples were obtained from two groups reared in separate sea pens and fed two different standard commercial diets. These diets have been anonymised but were manufactured respectively by BioMar and EWOS in 2018.
Six biological samples were taken from each fish, including muscle tissue for fatty acid profiling, gill tissue for host genomics, gut epithelia for host transcriptomics, gut epithelial cell scrapes for 16S metabarcoding and two gut content samples for metagenomics and metabolomics.
Approximate 100 mg distal gut content for each individual was sampled for metabolomics. Gut content for metabolomics was preserved at -80 degrees Celsius. All the sampling tools and equipment used for each sample were sterile.
Extraction
Gut content samples were cryo-homogenised in 25% water, 25% methanol and 50% dichloromethane in a 1:15 sample: solvent ratio (w:v). Homogenisation was carried out using an OMNI Bead Ruptor 24, using liquid nitrogen to keep homogenised samples below 0 degrees Celsius to minimise degradation of metabolites during extraction. Homogenates were centrifuged at 20,000 g (0 degrees Celsius) and the polar phase from all samples was concentrated using SpeedVac (ThermoFisher Scientific) and resuspended in 200 microL 5% methanol. Four procedural blanks were included in homogenisation. A volume of 100 microL of all samples was collected into a Quality Control sample used for normalisation to enhance the detection of metabolites.
Chromatography
Samples were measured on a nano-flow ultra-high pressure liquid chromatography tandem high-resolution mass spectrometry analysis.
Mass spectrometry
Metabolites were detected using a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher Scientific) operated in positive ion data-dependent acquisition mode.
Data Transformation
ThermoFisher Scientific UHPLC-Orbitrap-MS/MS RAW files were converted into mzML files using Proteo Wizard. For an increased deciphering of molecular spectres, MZmine2 was applied for mass detection of MS1 and MS2 spectres, followed by chromatogram detection and deconvolution. Subsequently, detected isotopes and features were grouped according to a tolerance of mass-charges (5 ppm for m/z) and retention time (6 sec.) and the features were further aligned according to retention time and m/z. Lastly, only features with an MS2 spectrum were kept for further substructural analysis and in silico analysis.
[doi:10.25345/C5T43JD5W]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Metabolomics ; Gut microbiome ; Atlantic salmon ; Aquaculture
Principal Investigators: (in alphabetical order) |
Jacob Rasmussen, University of Copenhagen, Denmark |
Submitting User: | JacobAgerbo |
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