#METABOLOMICS WORKBENCH gmartens_20221123_004553 DATATRACK_ID:3592 STUDY_ID:ST002358 ANALYSIS_ID:AN003850 PROJECT_ID:PR001514 VERSION 1 CREATED_ON November 23, 2022, 12:16 pm #PROJECT PR:PROJECT_TITLE Lipidomics of deep-diving pinniped brains PR:PROJECT_SUMMARY The brain of diving mammals such as the hooded seal (Cystophora cristata) PR:PROJECT_SUMMARY exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While PR:PROJECT_SUMMARY neurons of most terrestrial mammals suffer irreversible damage after only short PR:PROJECT_SUMMARY periods of hypoxia, in vitro experiments revealed that neurons of the hooded PR:PROJECT_SUMMARY seal show prolonged functional integrity even in severe hypoxia. As major PR:PROJECT_SUMMARY components of membranes, specific neuronal lipids of diving mammals could PR:PROJECT_SUMMARY contribute to the observed high hypoxia tolerance. Therefore, we analyzed the PR:PROJECT_SUMMARY brain lipidome of deep-diving pinnipeds (Cystophora cristata, Pagophilus PR:PROJECT_SUMMARY groenlandicus) in comparison to terrestrial (non-diving) relatives (Mustela PR:PROJECT_SUMMARY putorius furo, Mus musculus). Furthermore, lipid composition of C. cristata PR:PROJECT_SUMMARY brain tissue was analyzed that was exposed to hypoxia and reoxygenation in PR:PROJECT_SUMMARY vitro. PR:INSTITUTE University of Hamburg PR:LAST_NAME Martens PR:FIRST_NAME Gerrit Alexander PR:ADDRESS Martin-Luther-King-Platz 3, 20146 Hamburg, Germany PR:EMAIL gerrit.alexander.martens@uni-hamburg.de PR:PHONE +49 40 42838-3934 #STUDY ST:STUDY_TITLE Effect of hypoxia and reoxygenation on the juvenile hooded seal brain lipidome ST:STUDY_SUMMARY Brain samples from juvenile hooded seals (Cystophora cristata) were subjected to ST:STUDY_SUMMARY hypoxia and reoxygenation in vitro and lipid composition was compared. ST:INSTITUTE University of Hamburg ST:LAST_NAME Martens ST:FIRST_NAME Gerrit Alexander ST:ADDRESS Martin-Luther-King-Platz 3, 20146 Hamburg, Germany ST:EMAIL gerrit.alexander.martens@uni-hamburg.de ST:PHONE +49 40 42838-3934 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Cystophora cristata #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Ccr1-VC-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr1-VC-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-VC-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr1-VC-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-VC-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr1-VC-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-Hi-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr1-Hi-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-Hi-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr1-Hi-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-Hi-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr1-Hi-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-VC-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr2-VC-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-VC-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr2-VC-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-VC-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr2-VC-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-Hi-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr2-Hi-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-Hi-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr2-Hi-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-Hi-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr2-Hi-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-VC-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr3-VC-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-VC-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr3-VC-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-Hi-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr3-Hi-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-Hi-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr3-Hi-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-Hi-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr3-Hi-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-VC-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr4-VC-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-VC-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr4-VC-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-VC-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr4-VC-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-Hi-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr4-Hi-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-Hi-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr4-Hi-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-Hi-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr4-Hi-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr5-VC-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr5-VC-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr5-VC-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr5-VC-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr5-VC-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr5-VC-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr5-Hi-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr5-Hi-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr5-Hi-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr5-Hi-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr5-Hi-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr5-Hi-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr6-VC-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr6-VC-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr6-VC-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr6-VC-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr6-VC-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:visual cortex RAW_FILE_NAME=Ccr6-VC-HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr6-Hi-NO treatment:normoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr6-Hi-NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr6-Hi-HO treatment:hypoxia | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr6-Hi-HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr6-Hi-HONO treatment:reoxygenation | species:Cystophora cristata | age:juvenile | brain region:hippocampus RAW_FILE_NAME=Ccr6-Hi-HONO_xxx_xx_xxxxx.cdf #COLLECTION CO:COLLECTION_SUMMARY Hooded seals (Cystophora cristata) were captured for other scientific purposes CO:COLLECTION_SUMMARY in the pack ice of the Greenland Sea under permits from relevant Norwegian and CO:COLLECTION_SUMMARY Greenland authorities. The hooded seal pups were brought to UiT – The Arctic CO:COLLECTION_SUMMARY University of Norway, where they were maintained in a certified research animal CO:COLLECTION_SUMMARY facility in connection with other studies. At the termination of those CO:COLLECTION_SUMMARY experiments, the seals were euthanized (at age 1 year, respectively) in CO:COLLECTION_SUMMARY accordance with a permit issued by the National Animal Research Authority of CO:COLLECTION_SUMMARY Norway (permits no. 7247, 19305 and 22751): the seals were sedated by CO:COLLECTION_SUMMARY intramuscular injection of zolazepam/tiletamine (Zoletil Forte Vet., Virbac CO:COLLECTION_SUMMARY S.A., France; 1.5–2.0 mg per kg of body mass), then anaesthetized using an CO:COLLECTION_SUMMARY endotracheal tube to ventilate lungs with 2–3% isoflurane (Forene, Abbott, CO:COLLECTION_SUMMARY Germany) in air and, when fully anaesthetized, they were euthanized by CO:COLLECTION_SUMMARY exsanguination via the carotid arteries. All animal handling was in accordance CO:COLLECTION_SUMMARY with the Norwegian Animal Welfare Act and with approvals from the National CO:COLLECTION_SUMMARY Animal Research Authority of Norway (permits no. 7247, 19305 and 22751). Fresh CO:COLLECTION_SUMMARY visual cortex and hippocampus samples from hooded seal pups were minced and CO:COLLECTION_SUMMARY placed in cooled (4 °C) artificial cerebrospinal fluid (aCSF; 128 mM NaCl, 3 mM CO:COLLECTION_SUMMARY KCl, 1.5 mM CaCl2, 1 mM MgCl2, 24 mM NaHCO3, 0.5 mM NaH2PO4, 20 mM sucrose, 10 CO:COLLECTION_SUMMARY mM D-glucose) saturated with 95% O2−5% CO2 (normoxia) and further processed in CO:COLLECTION_SUMMARY vitro. CO:SAMPLE_TYPE Brain CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Samples in aCSF were adjusted to 34±0.5°C for at least 20 min. Hypoxia was TR:TREATMENT_SUMMARY introduced and maintained for 60 min after switching the gas supply to 95% N2 TR:TREATMENT_SUMMARY and 5% CO2, to mimic the diving brain. To simulate conditions when the seal TR:TREATMENT_SUMMARY surfaces after a dive, samples were exposed to hypoxia followed by 20 min return TR:TREATMENT_SUMMARY to normoxia. After treatment, hypoxia and reoxygenation samples were immediately TR:TREATMENT_SUMMARY frozen in liquid nitrogen. Samples that were kept under normoxia in aCSF for 80 TR:TREATMENT_SUMMARY min were used as controls. All samples were transferred to and stored at -80°C TR:TREATMENT_SUMMARY until later use. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The extraction protocol was performed slightly modified according to the method SP:SAMPLEPREP_SUMMARY of Bligh and Dyer (Bligh and Dyer 1959). About 20 mg sample was weighed into a SP:SAMPLEPREP_SUMMARY 2.0 ml reaction tube (Eppendorf, Hamburg, Germany). Two steel balls (3.6 mm), SP:SAMPLEPREP_SUMMARY 100 µl chloroform and 200 µl methanol were added to the sample. The mixture SP:SAMPLEPREP_SUMMARY was homogenized in a ball mill (1 min, 3.1 m/s Bead Ruptor 24, Omni SP:SAMPLEPREP_SUMMARY International IM, GA, USA)). Afterwards 200 µl water and 100 µl chloroform SP:SAMPLEPREP_SUMMARY were added and again processed in the ball mill (1 min, 3.1 m/s). The SP:SAMPLEPREP_SUMMARY homogenized sample was then centrifuged (20 min, 16.000xg, 5 °C, Sigma 3-16PK, SP:SAMPLEPREP_SUMMARY Sigma, Osterode, Germany). A quality control sample (QC) was prepared by SP:SAMPLEPREP_SUMMARY transferring 30 µl of each sample into a new vial. The organic chloroform phase SP:SAMPLEPREP_SUMMARY was directly used for measurement. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY LC experiments were carried out using a RP C-18 column (150 × 2.1 mm, 1.7 μm, CH:CHROMATOGRAPHY_SUMMARY Phenomenex, Aschaffenburg, Germany) together with a Dionex Ultimate 3000 UPLC CH:CHROMATOGRAPHY_SUMMARY system (Dionex, Idstein, Germany). The mobile phase consisted of water (solvent CH:CHROMATOGRAPHY_SUMMARY A) and mixture of acetonitrile and isopropanol (1:3, v/v) (solvent B). Both CH:CHROMATOGRAPHY_SUMMARY eluents contained 10 mMol/L ammonium formate for measurements in positive CH:CHROMATOGRAPHY_SUMMARY ionization mode and 0.02% acetic acid for measurements in negative ionization CH:CHROMATOGRAPHY_SUMMARY mode. The column oven was set at 50°C and the flow rate was 300 µL/min. The CH:CHROMATOGRAPHY_SUMMARY gradient elution was as follows: 55% B (0-2 minutes); 55% to 75% B (2-4 CH:CHROMATOGRAPHY_SUMMARY minutes); 75% to 100% B (4-18 minutes); 100% B (18-23 minutes), 55% B (23-24 CH:CHROMATOGRAPHY_SUMMARY minutes); 55% B (24-27 minutes). For measurements in positive ionization mode, 2 CH:CHROMATOGRAPHY_SUMMARY µL of the sample extracts were injected while for analyzes in negative CH:CHROMATOGRAPHY_SUMMARY ionization mode 8 µL were used. The samples were analyzed in randomized order, CH:CHROMATOGRAPHY_SUMMARY with one blank sample and one QC sample being measured after each of the five CH:CHROMATOGRAPHY_SUMMARY animal samples. The autosampler in which the samples were stored during the CH:CHROMATOGRAPHY_SUMMARY measurement was set to 4° C. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Dionex Ultimate 3000 UPLC system (Dionex, Idstein, Germany) CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 1.7um) CH:FLOW_GRADIENT 55% B (0-2 minutes); 55% to 75% B (2-4 minutes); 75% to 100% B (4-18 minutes); CH:FLOW_GRADIENT 100% B (18-23 minutes), 55% B (23-24 minutes); 55% B (24-27 minutes) CH:FLOW_RATE 300 µL/min CH:COLUMN_TEMPERATURE 50°C CH:SOLVENT_A water CH:SOLVENT_B acetonitrile and isopropanol (1:3, v/v) CH:INTERNAL_STANDARD 10 mMol/L ammonium formate for measurements in positive ionization mode and CH:INTERNAL_STANDARD 0.02% acetic acid for measurements in negative ionization mode CH:SAMPLE_INJECTION 2 µL of sample extracts were injected for measurements in positive ionization CH:SAMPLE_INJECTION mode and 2µl of the sample extracts were injected for analyzes in negative CH:SAMPLE_INJECTION ionization mode #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Daltonics maXis 3G MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The data were recorded at 1 Hz over a mass range of m/z 80-1100. Further MS:MS_COMMENTS parameters were: end plate offset -500 V, capillary -4500 V, nebulizer pressure MS:MS_COMMENTS 4.0 bar, dry gas 9.0 L/min at 200 °C dry temperature. At the beginning of the MS:MS_COMMENTS measurements, the mass spectrometer was calibrated using sodium formate MS:MS_COMMENTS clusters. At the end of each sample run, a further calibration was carried out MS:MS_COMMENTS using the cluster solutions. Acquired experimental mass spectra were MS:MS_COMMENTS recalibrated with Bruker Data Analysis Software 4.2 (Bruker Daltonics, Bremen, MS:MS_COMMENTS Germany) using the mentioned sodium formate clusters. Afterwards, data were MS:MS_COMMENTS exported to netCDF file format. Data preprocessing was performed with R package MS:MS_COMMENTS xcms 3.6.2 (Smith et al. 2006) in R version 3.6.3 (R Core Team 2021). Parameters MS:MS_COMMENTS for processing were optimized based on existing tools and scripts (Libiseller et MS:MS_COMMENTS al. 2015; Manier et al. 2019). After reading in recalibrated netCDF files, MS:MS_COMMENTS features were detected with findChromPeaks function and CentWaveParam (peakwidth MS:MS_COMMENTS = c(10, 40), ppm = 20, snthresh = 10, mzdiff = 0.015, prefilter = c(0, 0), noise MS:MS_COMMENTS = 0)). Retention time was corrected with adjustRtime function and ObiwarpParam MS:MS_COMMENTS (binSize = 1.0). Feature correspondence was achieved with groupChromPeaks MS:MS_COMMENTS function and PeakDensityParam (sampleGroups = xdata$sample_group, bw = 1) as MS:MS_COMMENTS well as missing value imputation with fillChromPeaks function with MS:MS_COMMENTS FillChromPeaksParam (fixedRt = ChromPeakwidth/2)). ChromPeakwidth was calculated MS:MS_COMMENTS as average peak width of detected chromatographic peaks. Adducts and isotopes of MS:MS_COMMENTS features were annotated using R package CAMERA 1.40.0 (Kuhl et al. 2012). MS:MS_COMMENTS Features in the QC samples with a relative standard deviation over 30%, blank MS:MS_COMMENTS intensity contribution over 10% and QC sample count below 60% were removed MS:MS_COMMENTS before further statistical analysis. MS:CAPILLARY_VOLTAGE -4500 V MS:DRY_GAS_FLOW 9.0 L/min MS:DRY_GAS_TEMP 200 °C MS:NEBULIZER 4.0 bar MS:MS_RESULTS_FILE ST002358_AN003850_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END