Summary of Study ST002358

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 PR001514. The data can be accessed directly via it's Project DOI: 10.21228/M80H6B This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002358
Study TitleEffect of hypoxia and reoxygenation on the juvenile hooded seal brain lipidome
Study SummaryBrain samples from juvenile hooded seals (Cystophora cristata) were subjected to hypoxia and reoxygenation in vitro and lipid composition was compared.
Institute
University of Hamburg
Last NameMartens
First NameGerrit Alexander
AddressMartin-Luther-King-Platz 3, 20146 Hamburg, Germany
Emailgerrit.alexander.martens@uni-hamburg.de
Phone+49 40 42838-3934
Submit Date2022-11-23
Raw Data AvailableYes
Raw Data File Type(s)cdf
Analysis Type DetailLC-MS
Release Date2023-04-12
Release Version1
Gerrit Alexander Martens Gerrit Alexander Martens
https://dx.doi.org/10.21228/M80H6B
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001514
Project DOI:doi: 10.21228/M80H6B
Project Title:Lipidomics of deep-diving pinniped brains
Project Summary:The brain of diving mammals such as the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments revealed that neurons of the hooded seal show prolonged functional integrity even in severe hypoxia. As major components of membranes, specific neuronal lipids of diving mammals could contribute to the observed high hypoxia tolerance. Therefore, we analyzed the brain lipidome of deep-diving pinnipeds (Cystophora cristata, Pagophilus groenlandicus) in comparison to terrestrial (non-diving) relatives (Mustela putorius furo, Mus musculus). Furthermore, lipid composition of C. cristata brain tissue was analyzed that was exposed to hypoxia and reoxygenation in vitro.
Institute:University of Hamburg
Last Name:Martens
First Name:Gerrit Alexander
Address:Martin-Luther-King-Platz 3, 20146 Hamburg, Germany
Email:gerrit.alexander.martens@uni-hamburg.de
Phone:+49 40 42838-3934

Subject:

Subject ID:SU002447
Subject Type:Mammal
Subject Species:Cystophora cristata
Species Group:Mammals

Factors:

Subject type: Mammal; Subject species: Cystophora cristata (Factor headings shown in green)

mb_sample_id local_sample_id treatment species age brain region
SA236929Ccr3-Hi-HOhypoxia Cystophora cristata juvenile hippocampus
SA236930Ccr2-Hi-HOhypoxia Cystophora cristata juvenile hippocampus
SA236931Ccr6-Hi-HOhypoxia Cystophora cristata juvenile hippocampus
SA236932Ccr4-Hi-HOhypoxia Cystophora cristata juvenile hippocampus
SA236933Ccr1-Hi-HOhypoxia Cystophora cristata juvenile hippocampus
SA236934Ccr5-Hi-HOhypoxia Cystophora cristata juvenile hippocampus
SA236935Ccr5-VC-HOhypoxia Cystophora cristata juvenile visual cortex
SA236936Ccr4-VC-HOhypoxia Cystophora cristata juvenile visual cortex
SA236937Ccr1-VC-HOhypoxia Cystophora cristata juvenile visual cortex
SA236938Ccr2-VC-HOhypoxia Cystophora cristata juvenile visual cortex
SA236939Ccr6-VC-HOhypoxia Cystophora cristata juvenile visual cortex
SA236940Ccr3-Hi-NOnormoxia Cystophora cristata juvenile hippocampus
SA236941Ccr2-Hi-NOnormoxia Cystophora cristata juvenile hippocampus
SA236942Ccr1-Hi-NOnormoxia Cystophora cristata juvenile hippocampus
SA236943Ccr6-Hi-NOnormoxia Cystophora cristata juvenile hippocampus
SA236944Ccr4-Hi-NOnormoxia Cystophora cristata juvenile hippocampus
SA236945Ccr5-Hi-NOnormoxia Cystophora cristata juvenile hippocampus
SA236946Ccr6-VC-NOnormoxia Cystophora cristata juvenile visual cortex
SA236947Ccr5-VC-NOnormoxia Cystophora cristata juvenile visual cortex
SA236948Ccr4-VC-NOnormoxia Cystophora cristata juvenile visual cortex
SA236949Ccr2-VC-NOnormoxia Cystophora cristata juvenile visual cortex
SA236950Ccr3-VC-NOnormoxia Cystophora cristata juvenile visual cortex
SA236951Ccr1-VC-NOnormoxia Cystophora cristata juvenile visual cortex
SA236952Ccr2-Hi-HONOreoxygenation Cystophora cristata juvenile hippocampus
SA236953Ccr6-Hi-HONOreoxygenation Cystophora cristata juvenile hippocampus
SA236954Ccr1-Hi-HONOreoxygenation Cystophora cristata juvenile hippocampus
SA236955Ccr5-Hi-HONOreoxygenation Cystophora cristata juvenile hippocampus
SA236956Ccr3-Hi-HONOreoxygenation Cystophora cristata juvenile hippocampus
SA236957Ccr4-Hi-HONOreoxygenation Cystophora cristata juvenile hippocampus
SA236958Ccr1-VC-HONOreoxygenation Cystophora cristata juvenile visual cortex
SA236959Ccr5-VC-HONOreoxygenation Cystophora cristata juvenile visual cortex
SA236960Ccr6-VC-HONOreoxygenation Cystophora cristata juvenile visual cortex
SA236961Ccr3-VC-HONOreoxygenation Cystophora cristata juvenile visual cortex
SA236962Ccr2-VC-HONOreoxygenation Cystophora cristata juvenile visual cortex
SA236963Ccr4-VC-HONOreoxygenation Cystophora cristata juvenile visual cortex
Showing results 1 to 35 of 35

Collection:

Collection ID:CO002440
Collection Summary:Hooded seals (Cystophora cristata) were captured for other scientific purposes in the pack ice of the Greenland Sea under permits from relevant Norwegian and Greenland authorities. The hooded seal pups were brought to UiT – The Arctic University of Norway, where they were maintained in a certified research animal facility in connection with other studies. At the termination of those experiments, the seals were euthanized (at age 1 year, respectively) in accordance with a permit issued by the National Animal Research Authority of Norway (permits no. 7247, 19305 and 22751): the seals were sedated by intramuscular injection of zolazepam/tiletamine (Zoletil Forte Vet., Virbac S.A., France; 1.5–2.0 mg per kg of body mass), then anaesthetized using an endotracheal tube to ventilate lungs with 2–3% isoflurane (Forene, Abbott, Germany) in air and, when fully anaesthetized, they were euthanized by exsanguination via the carotid arteries. All animal handling was in accordance with the Norwegian Animal Welfare Act and with approvals from the National Animal Research Authority of Norway (permits no. 7247, 19305 and 22751). Fresh visual cortex and hippocampus samples from hooded seal pups were minced and placed in cooled (4 °C) artificial cerebrospinal fluid (aCSF; 128 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 24 mM NaHCO3, 0.5 mM NaH2PO4, 20 mM sucrose, 10 mM D-glucose) saturated with 95% O2−5% CO2 (normoxia) and further processed in vitro.
Sample Type:Brain
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002459
Treatment Summary:Samples in aCSF were adjusted to 34±0.5°C for at least 20 min. Hypoxia was introduced and maintained for 60 min after switching the gas supply to 95% N2 and 5% CO2, to mimic the diving brain. To simulate conditions when the seal surfaces after a dive, samples were exposed to hypoxia followed by 20 min return to normoxia. After treatment, hypoxia and reoxygenation samples were immediately frozen in liquid nitrogen. Samples that were kept under normoxia in aCSF for 80 min were used as controls. All samples were transferred to and stored at -80°C until later use.

Sample Preparation:

Sampleprep ID:SP002453
Sampleprep Summary:The extraction protocol was performed slightly modified according to the method of Bligh and Dyer (Bligh and Dyer 1959). About 20 mg sample was weighed into a 2.0 ml reaction tube (Eppendorf, Hamburg, Germany). Two steel balls (3.6 mm), 100 µl chloroform and 200 µl methanol were added to the sample. The mixture was homogenized in a ball mill (1 min, 3.1 m/s Bead Ruptor 24, Omni International IM, GA, USA)). Afterwards 200 µl water and 100 µl chloroform were added and again processed in the ball mill (1 min, 3.1 m/s). The homogenized sample was then centrifuged (20 min, 16.000xg, 5 °C, Sigma 3-16PK, Sigma, Osterode, Germany). A quality control sample (QC) was prepared by transferring 30 µl of each sample into a new vial. The organic chloroform phase was directly used for measurement.

Combined analysis:

Analysis ID AN003850 AN003851
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Dionex Ultimate 3000 UPLC system (Dionex, Idstein, Germany) Dionex Ultimate 3000 UPLC system (Dionex, Idstein, Germany)
Column Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Bruker Daltonics maXis 3G Bruker Daltonics maXis 3G
Ion Mode POSITIVE NEGATIVE
Units Peak Area Peak Area

Chromatography:

Chromatography ID:CH002850
Chromatography Summary:LC experiments were carried out using a RP C-18 column (150 × 2.1 mm, 1.7 μm, Phenomenex, Aschaffenburg, Germany) together with a Dionex Ultimate 3000 UPLC system (Dionex, Idstein, Germany). The mobile phase consisted of water (solvent A) and mixture of acetonitrile and isopropanol (1:3, v/v) (solvent B). Both eluents contained 10 mMol/L ammonium formate for measurements in positive ionization mode and 0.02% acetic acid for measurements in negative ionization mode. The column oven was set at 50°C and the flow rate was 300 µL/min. The gradient elution was as follows: 55% B (0-2 minutes); 55% to 75% B (2-4 minutes); 75% to 100% B (4-18 minutes); 100% B (18-23 minutes), 55% B (23-24 minutes); 55% B (24-27 minutes). For measurements in positive ionization mode, 2 µL of the sample extracts were injected while for analyzes in negative ionization mode 8 µL were used. The samples were analyzed in randomized order, with one blank sample and one QC sample being measured after each of the five animal samples. The autosampler in which the samples were stored during the measurement was set to 4° C.
Instrument Name:Dionex Ultimate 3000 UPLC system (Dionex, Idstein, Germany)
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:50°C
Flow Gradient:55% B (0-2 minutes); 55% to 75% B (2-4 minutes); 75% to 100% B (4-18 minutes); 100% B (18-23 minutes), 55% B (23-24 minutes); 55% B (24-27 minutes)
Flow Rate:300 µL/min
Internal Standard:10 mMol/L ammonium formate for measurements in positive ionization mode and 0.02% acetic acid for measurements in negative ionization mode
Sample Injection:2 µL of sample extracts were injected for measurements in positive ionization mode and 2µl of the sample extracts were injected for analyzes in negative ionization mode
Solvent A:100% water
Solvent B:25% acetonitrile/75% isopropanol
Chromatography Type:Reversed phase

MS:

MS ID:MS003591
Analysis ID:AN003850
Instrument Name:Bruker Daltonics maXis 3G
Instrument Type:QTOF
MS Type:ESI
MS Comments:The data were recorded at 1 Hz over a mass range of m/z 80-1100. Further parameters were: end plate offset -500 V, capillary -4500 V, nebulizer pressure 4.0 bar, dry gas 9.0 L/min at 200 °C dry temperature. At the beginning of the measurements, the mass spectrometer was calibrated using sodium formate clusters. At the end of each sample run, a further calibration was carried out using the cluster solutions. Acquired experimental mass spectra were recalibrated with Bruker Data Analysis Software 4.2 (Bruker Daltonics, Bremen, Germany) using the mentioned sodium formate clusters. Afterwards, data were exported to netCDF file format. Data preprocessing was performed with R package xcms 3.6.2 (Smith et al. 2006) in R version 3.6.3 (R Core Team 2021). Parameters for processing were optimized based on existing tools and scripts (Libiseller et al. 2015; Manier et al. 2019). After reading in recalibrated netCDF files, features were detected with findChromPeaks function and CentWaveParam (peakwidth = c(10, 40), ppm = 20, snthresh = 10, mzdiff = 0.015, prefilter = c(0, 0), noise = 0)). Retention time was corrected with adjustRtime function and ObiwarpParam (binSize = 1.0). Feature correspondence was achieved with groupChromPeaks function and PeakDensityParam (sampleGroups = xdata$sample_group, bw = 1) as well as missing value imputation with fillChromPeaks function with FillChromPeaksParam (fixedRt = ChromPeakwidth/2)). ChromPeakwidth was calculated as average peak width of detected chromatographic peaks. Adducts and isotopes of features were annotated using R package CAMERA 1.40.0 (Kuhl et al. 2012). Features in the QC samples with a relative standard deviation over 30%, blank intensity contribution over 10% and QC sample count below 60% were removed before further statistical analysis.
Ion Mode:POSITIVE
Capillary Voltage:-4500 V
Dry Gas Flow:9.0 L/min
Dry Gas Temp:200 °C
Nebulizer:4.0 bar
  
MS ID:MS003592
Analysis ID:AN003851
Instrument Name:Bruker Daltonics maXis 3G
Instrument Type:QTOF
MS Type:ESI
MS Comments:The data were recorded at 1 Hz over a mass range of m/z 80-1100. Further parameters were: end plate offset -500 V, capillary +4500 V, nebulizer pressure 4.0 bar, dry gas 9.0 L/min at 200 °C dry temperature. At the beginning of the measurements, the mass spectrometer was calibrated using sodium acetate clusters. At the end of each sample run, a further calibration was carried out using the cluster solutions. Acquired experimental mass spectra were recalibrated with Bruker Data Analysis Software 4.2 (Bruker Daltonics, Bremen, Germany) using the mentioned sodium acetate clusters. Afterwards, data were exported to netCDF file format. Data preprocessing was performed with R package xcms 3.6.2 (Smith et al. 2006) in R version 3.6.3 (R Core Team 2021). Parameters for processing were optimized based on existing tools and scripts (Libiseller et al. 2015; Manier et al. 2019). After reading in recalibrated netCDF files, features were detected with findChromPeaks function and CentWaveParam (peakwidth = c(10, 40), ppm = 20, snthresh = 10, mzdiff = 0.015, prefilter = c(0, 0), noise = 0)). Retention time was corrected with adjustRtime function and ObiwarpParam (binSize = 1.0). Feature correspondence was achieved with groupChromPeaks function and PeakDensityParam (sampleGroups = xdata$sample_group, bw = 1) as well as missing value imputation with fillChromPeaks function with FillChromPeaksParam (fixedRt = ChromPeakwidth/2)). ChromPeakwidth was calculated as average peak width of detected chromatographic peaks. Adducts and isotopes of features were annotated using R package CAMERA 1.40.0 (Kuhl et al. 2012). Features in the QC samples with a relative standard deviation over 30%, blank intensity contribution over 10% and QC sample count below 60% were removed before further statistical analysis.
Ion Mode:NEGATIVE
Capillary Voltage:+4500 V
Dry Gas Flow:9.0 L/min
Dry Gas Temp:200 °C
Nebulizer:4.0 bar
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