#METABOLOMICS WORKBENCH gmartens_20221122_010813 DATATRACK_ID:3590 STUDY_ID:ST002357 ANALYSIS_ID:AN003849 PROJECT_ID:PR001514 VERSION 1 CREATED_ON November 23, 2022, 12:17 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 Comparative lipidomics study of brain samples from deep-diving pinnipeds and ST:STUDY_TITLE terrestrial (non-diving) relatives ST:STUDY_SUMMARY Brain samples from deep-diving pinnipeds (Cystophora cristata (n=10), Pagophilus ST:STUDY_SUMMARY groenlandicus (n=6)) were compared to terrestrial (non-diving) relatives ST:STUDY_SUMMARY (Mustela putorius furo (n=4), Mus musculus (n=9)). 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/Pagophilus groenlandicus/Mustela putorius furo/Mus musculus #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 - Ccr_adu1 species:Cystophora cristata | age:adult RAW_FILE_NAME=Ccr_adu1_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_adu2 species:Cystophora cristata | age:adult RAW_FILE_NAME=Ccr_adu2_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_adu3 species:Cystophora cristata | age:adult RAW_FILE_NAME=Ccr_adu3_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_adu4 species:Cystophora cristata | age:adult RAW_FILE_NAME=Ccr_adu4_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_adu5 species:Cystophora cristata | age:adult RAW_FILE_NAME=Ccr_adu5_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_adu6 species:Cystophora cristata | age:adult RAW_FILE_NAME=Ccr_adu6_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_juv1 species:Cystophora cristata | age:juvenile RAW_FILE_NAME=Ccr_juv1_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_juv2 species:Cystophora cristata | age:juvenile RAW_FILE_NAME=Ccr_juv2_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_juv3 species:Cystophora cristata | age:juvenile RAW_FILE_NAME=Ccr_juv3_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr_juv4 species:Cystophora cristata | age:juvenile RAW_FILE_NAME=Ccr_juv4_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Pgr1 species:Pagophilus groenlandicus | age:adult RAW_FILE_NAME=Pgr1_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Pgr2 species:Pagophilus groenlandicus | age:adult RAW_FILE_NAME=Pgr2_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Pgr3 species:Pagophilus groenlandicus | age:adult RAW_FILE_NAME=Pgr3_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Pgr4 species:Pagophilus groenlandicus | age:adult RAW_FILE_NAME=Pgr4_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Pgr5 species:Pagophilus groenlandicus | age:adult RAW_FILE_NAME=Pgr5_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Pgr6 species:Pagophilus groenlandicus | age:adult RAW_FILE_NAME=Pgr6_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mpu1 species:Mustela putorius furo | age:adult RAW_FILE_NAME=Mpu1_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mpu2 species:Mustela putorius furo | age:adult RAW_FILE_NAME=Mpu2_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mpu3 species:Mustela putorius furo | age:adult RAW_FILE_NAME=Mpu3_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mpu4 species:Mustela putorius furo | age:adult RAW_FILE_NAME=Mpu4_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu1 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu1_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu2 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu2_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu3 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu3_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu4 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu4_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu5 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu5_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu6 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu6_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu7 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu7_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu8 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu8_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Mmu9 species:Mus musculus | age:adult RAW_FILE_NAME=Mmu9_xxx_xx_xxxxx.cdf #COLLECTION CO:COLLECTION_SUMMARY Hooded seals (Cystophora cristata) and harp seals (Pagophilus groenlandicus) CO:COLLECTION_SUMMARY were captured for other scientific purposes in the pack ice of the Greenland Sea CO:COLLECTION_SUMMARY under permits from relevant Norwegian and Greenland authorities. The hooded seal CO:COLLECTION_SUMMARY pups were brought to UiT – The Arctic University of Norway, where they were CO:COLLECTION_SUMMARY maintained in a certified research animal facility in connection with other CO:COLLECTION_SUMMARY studies. At the termination of those experiments, the seals were euthanized (at CO:COLLECTION_SUMMARY age 1 year, respectively) in accordance with a permit issued by the National CO:COLLECTION_SUMMARY Animal Research Authority of Norway (permits no. 7247, 19305 and 22751): the CO:COLLECTION_SUMMARY seals were sedated by intramuscular injection of zolazepam/tiletamine (Zoletil CO:COLLECTION_SUMMARY Forte Vet., Virbac S.A., France; 1.5–2.0 mg per kg of body mass), then CO:COLLECTION_SUMMARY anaesthetized using an endotracheal tube to ventilate lungs with 2–3% CO:COLLECTION_SUMMARY isoflurane (Forene, Abbott, Germany) in air and, when fully anaesthetized, they CO:COLLECTION_SUMMARY were euthanized by exsanguination via the carotid arteries. The adult hooded and CO:COLLECTION_SUMMARY harp seals were euthanized immediately following capture, by sedation with CO:COLLECTION_SUMMARY intramuscular injection of zolazepam/tiletamine (1.5–2.0 mg per kg of body CO:COLLECTION_SUMMARY mass), followed by catheterization of the extradural intravertebral vein and CO:COLLECTION_SUMMARY i.v. injection of an overdose of pentobarbital (Euthasol vet., Le Vet B.V., CO:COLLECTION_SUMMARY Netherlands; 30 mg per kg of body mass). 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). Samples CO:COLLECTION_SUMMARY of adult female ferrets were received from the animal facilities of the CO:COLLECTION_SUMMARY University Medical Center Hamburg-Eppendorf (UKE) Germany. The ferrets were CO:COLLECTION_SUMMARY killed at the UKE in deep anesthesia (Ketamin/Domitor) with an overdose of CO:COLLECTION_SUMMARY pentobarbital and brain tissues were sampled by the facility’s veterinarians. CO:COLLECTION_SUMMARY Adult female mice (C57BL/6) were a gift by Prof. Dr. Christian Lohr (University CO:COLLECTION_SUMMARY of Hamburg, Hamburg, Germany) and were anaesthetized with 1 ml isoflurane CO:COLLECTION_SUMMARY (Forene, Abbott, Germany) in a chamber and decapitated. All animals were handled CO:COLLECTION_SUMMARY according to the EU Directive 63 (Directive 2010/63/EU). Fresh brain tissue of CO:COLLECTION_SUMMARY all animals was frozen in liquid nitrogen and transferred to -80 °C to be CO:COLLECTION_SUMMARY stored for subsequent analysis. CO:SAMPLE_TYPE Brain CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY NA #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 NEGATIVE 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 acetate 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 acetate 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 ST002357_AN003849_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END