#METABOLOMICS WORKBENCH gmartens_20221123_030335 DATATRACK_ID:3593 STUDY_ID:ST002359 ANALYSIS_ID:AN003852 PROJECT_ID:PR001514 VERSION 1 CREATED_ON November 23, 2022, 12:14 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 adult hooded seal brain lipidome ST:STUDY_SUMMARY Brain samples from adult 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 - Ccr1-1_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:1 RAW_FILE_NAME=Ccr1-1_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-2_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:2 RAW_FILE_NAME=Ccr1-2_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-3_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:3 RAW_FILE_NAME=Ccr1-3_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-1_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:1 RAW_FILE_NAME=Ccr2-1_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-2_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:2 RAW_FILE_NAME=Ccr2-2_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-3_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:3 RAW_FILE_NAME=Ccr2-3_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-1_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:1 RAW_FILE_NAME=Ccr3-1_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-2_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:2 RAW_FILE_NAME=Ccr3-2_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-3_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:3 RAW_FILE_NAME=Ccr3-3_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-1_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:1 RAW_FILE_NAME=Ccr4-1_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-2_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:2 RAW_FILE_NAME=Ccr4-2_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-3_VC_NO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:normoxia | replicate:3 RAW_FILE_NAME=Ccr4-3_VC_NO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-1_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:1 RAW_FILE_NAME=Ccr1-1_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-2_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:2 RAW_FILE_NAME=Ccr1-2_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-3_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:3 RAW_FILE_NAME=Ccr1-3_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-1_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:1 RAW_FILE_NAME=Ccr2-1_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-2_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:2 RAW_FILE_NAME=Ccr2-2_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-3_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:3 RAW_FILE_NAME=Ccr2-3_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-1_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:1 RAW_FILE_NAME=Ccr3-1_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-2_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:2 RAW_FILE_NAME=Ccr3-2_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-3_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:3 RAW_FILE_NAME=Ccr3-3_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-1_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:1 RAW_FILE_NAME=Ccr4-1_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-2_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:2 RAW_FILE_NAME=Ccr4-2_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-3_VC_HO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:hypoxia | replicate:3 RAW_FILE_NAME=Ccr4-3_VC_HO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-1_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:1 RAW_FILE_NAME=Ccr1-1_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-2_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:2 RAW_FILE_NAME=Ccr1-2_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr1-3_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:3 RAW_FILE_NAME=Ccr1-3_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-1_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:1 RAW_FILE_NAME=Ccr2-1_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-2_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:2 RAW_FILE_NAME=Ccr2-2_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr2-3_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:3 RAW_FILE_NAME=Ccr2-3_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-1_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:1 RAW_FILE_NAME=Ccr3-1_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-2_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:2 RAW_FILE_NAME=Ccr3-2_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr3-3_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:3 RAW_FILE_NAME=Ccr3-3_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-1_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:1 RAW_FILE_NAME=Ccr4-1_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-2_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:2 RAW_FILE_NAME=Ccr4-2_VC_HONO_xxx_xx_xxxxx.cdf SUBJECT_SAMPLE_FACTORS - Ccr4-3_VC_HONO species:Cystophora cristata | age:adult | brain region:visual cortex | treatment:reoxygenation | replicate:3 RAW_FILE_NAME=Ccr4-3_VC_HONO_xxx_xx_xxxxx.cdf #COLLECTION CO:COLLECTION_SUMMARY Hooded seals (Cystophora cristata) were captured in the pack ice of the CO:COLLECTION_SUMMARY Greenland Sea under permits from relevant Norwegian and Greenland authorities. CO:COLLECTION_SUMMARY The adult hooded seals were euthanized immediately following capture, by CO:COLLECTION_SUMMARY sedation with intramuscular injection of zolazepam/tiletamine (1.5–2.0 mg per CO:COLLECTION_SUMMARY kg of body mass), followed by catheterization of the extradural intravertebral CO:COLLECTION_SUMMARY vein and i.v. injection of an overdose of pentobarbital (Euthasol vet., Le Vet CO:COLLECTION_SUMMARY B.V., Netherlands; 30 mg per kg of body mass). All animal handling was in CO:COLLECTION_SUMMARY accordance with the Norwegian Animal Welfare Act and with approvals from the CO:COLLECTION_SUMMARY National Animal Research Authority of Norway (permits no. 7247, 19305 and CO:COLLECTION_SUMMARY 22751). Fresh visual cortex samples from hooded seal adults 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. Because of limited sample number, technical SP:SAMPLEPREP_SUMMARY triplicates of each sample were measured. #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 ST002359_AN003852_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END