Summary of Study ST002359
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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST002359 |
| Study Title | Effect of hypoxia and reoxygenation on the adult hooded seal brain lipidome |
| Study Summary | Brain samples from adult hooded seals (Cystophora cristata) were subjected to hypoxia and reoxygenation in vitro and lipid composition was compared. |
| Institute | University of Hamburg |
| Last Name | Martens |
| First Name | Gerrit Alexander |
| Address | Martin-Luther-King-Platz 3, 20146 Hamburg, Germany |
| gerrit.alexander.martens@uni-hamburg.de | |
| Phone | +49 40 42838-3934 |
| Submit Date | 2022-11-23 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | cdf |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-04-12 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | SU002448 |
| 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 | species | age | brain region | treatment | replicate |
|---|---|---|---|---|---|---|
| SA236964 | Ccr2-1_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 1 |
| SA236965 | Ccr4-1_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 1 |
| SA236966 | Ccr1-1_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 1 |
| SA236967 | Ccr3-1_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 1 |
| SA236968 | Ccr2-2_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 2 |
| SA236969 | Ccr1-2_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 2 |
| SA236970 | Ccr3-2_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 2 |
| SA236971 | Ccr4-2_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 2 |
| SA236972 | Ccr1-3_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 3 |
| SA236973 | Ccr2-3_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 3 |
| SA236974 | Ccr4-3_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 3 |
| SA236975 | Ccr3-3_VC_HO | Cystophora cristata | adult | visual cortex | hypoxia | 3 |
| SA236976 | Ccr4-1_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 1 |
| SA236977 | Ccr1-1_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 1 |
| SA236978 | Ccr2-1_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 1 |
| SA236979 | Ccr3-1_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 1 |
| SA236980 | Ccr2-2_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 2 |
| SA236981 | Ccr4-2_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 2 |
| SA236982 | Ccr1-2_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 2 |
| SA236983 | Ccr3-2_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 2 |
| SA236984 | Ccr3-3_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 3 |
| SA236985 | Ccr4-3_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 3 |
| SA236986 | Ccr1-3_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 3 |
| SA236987 | Ccr2-3_VC_NO | Cystophora cristata | adult | visual cortex | normoxia | 3 |
| SA236988 | Ccr4-1_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 1 |
| SA236989 | Ccr3-1_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 1 |
| SA236990 | Ccr1-1_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 1 |
| SA236991 | Ccr2-1_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 1 |
| SA236992 | Ccr2-2_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 2 |
| SA236993 | Ccr1-2_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 2 |
| SA236994 | Ccr3-2_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 2 |
| SA236995 | Ccr4-2_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 2 |
| SA236996 | Ccr4-3_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 3 |
| SA236997 | Ccr3-3_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 3 |
| SA236998 | Ccr2-3_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 3 |
| SA236999 | Ccr1-3_VC_HONO | Cystophora cristata | adult | visual cortex | reoxygenation | 3 |
| Showing results 1 to 36 of 36 |
Collection:
| Collection ID: | CO002441 |
| Collection Summary: | Hooded seals (Cystophora cristata) were captured in the pack ice of the Greenland Sea under permits from relevant Norwegian and Greenland authorities. The adult hooded seals were euthanized immediately following capture, by sedation with intramuscular injection of zolazepam/tiletamine (1.5–2.0 mg per kg of body mass), followed by catheterization of the extradural intravertebral vein and i.v. injection of an overdose of pentobarbital (Euthasol vet., Le Vet B.V., Netherlands; 30 mg per kg of body mass). 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 samples from hooded seal adults 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: | TR002460 |
| 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: | SP002454 |
| 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. Because of limited sample number, technical triplicates of each sample were measured. |
Combined analysis:
| Analysis ID | AN003852 | AN003853 |
|---|---|---|
| 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: | CH002851 |
| 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: | MS003593 |
| Analysis ID: | AN003852 |
| 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: | MS003594 |
| Analysis ID: | AN003853 |
| 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 |
