#METABOLOMICS WORKBENCH Lmingram_20200730_193019 DATATRACK_ID:2107 STUDY_ID:ST001525 ANALYSIS_ID:AN002546 PROJECT_ID:PR001026 VERSION 1 CREATED_ON November 11, 2020, 2:10 pm #PROJECT PR:PROJECT_TITLE Perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) PR:PROJECT_TITLE alter the blood lipidome and the hepatic proteome in a murine model of PR:PROJECT_TITLE diet-induced obesity PR:PROJECT_TYPE Lipidomics PR:PROJECT_SUMMARY Perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) PR:PROJECT_SUMMARY alter the blood lipidome and the hepatic proteome in a murine model of PR:PROJECT_SUMMARY diet-induced obesity PR:INSTITUTE University of Rhode Island;University of Georgia PR:DEPARTMENT Pharmaceutical and Biomedical Sciences PR:LABORATORY Cummings/Slitt PR:LAST_NAME Ingram;Cummings PR:FIRST_NAME Lishann;Brian PR:ADDRESS 250 West Green Street PR:EMAIL ingram@carnegiescience.edu;briansc@uga.edu PR:PHONE 706-542-3792 PR:FUNDING_SOURCE NIEHS;DOD #STUDY ST:STUDY_TITLE Perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) ST:STUDY_TITLE alter the blood lipidome and the hepatic proteome in a murine model of ST:STUDY_TITLE diet-induced obesity ST:STUDY_SUMMARY Perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) ST:STUDY_SUMMARY alter the blood lipidome and the hepatic proteome in a murine model of ST:STUDY_SUMMARY diet-induced obesity ST:INSTITUTE University of Rhode Island;University of Georgia ST:DEPARTMENT Pharmaceutical and Biomedical Sciences ST:LABORATORY Cummings/Slitt ST:LAST_NAME Ingram ST:FIRST_NAME Lishann ST:ADDRESS 250 West Green Street Athens, GA 30605 ST:EMAIL ingram@carnegiescience.edu ST:PHONE 706-542-3792 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:AGE_OR_AGE_RANGE 8 weeks SU:GENDER Male and female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs (Bar Harbor, ME USA) #FACTORS #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 - S1 Diet:LFD | Treatment:none RAW_FILE_NAME=rf1 SUBJECT_SAMPLE_FACTORS - S2 Diet:LFD | Treatment:none RAW_FILE_NAME=rf2 SUBJECT_SAMPLE_FACTORS - S3 Diet:LFD | Treatment:none RAW_FILE_NAME=rf3 SUBJECT_SAMPLE_FACTORS - S4 Diet:LFD | Treatment:none RAW_FILE_NAME=rf4 SUBJECT_SAMPLE_FACTORS - S5 Diet:LFD | Treatment:none RAW_FILE_NAME=rf5 SUBJECT_SAMPLE_FACTORS - S6 Diet:LFD | Treatment:none RAW_FILE_NAME=rf6 SUBJECT_SAMPLE_FACTORS - S7 Diet:LFD | Treatment:PFOS RAW_FILE_NAME=rf7 SUBJECT_SAMPLE_FACTORS - S8 Diet:LFD | Treatment:PFOS RAW_FILE_NAME=rf8 SUBJECT_SAMPLE_FACTORS - S9 Diet:LFD | Treatment:PFOS RAW_FILE_NAME=rf9 SUBJECT_SAMPLE_FACTORS - S10 Diet:LFD | Treatment:PFOS RAW_FILE_NAME=rf10 SUBJECT_SAMPLE_FACTORS - S11 Diet:LFD | Treatment:PFOS RAW_FILE_NAME=rf11 SUBJECT_SAMPLE_FACTORS - S12 Diet:LFD | Treatment:PFOS RAW_FILE_NAME=rf12 SUBJECT_SAMPLE_FACTORS - S13 Diet:LFD | Treatment:PFHxS RAW_FILE_NAME=rf13 SUBJECT_SAMPLE_FACTORS - S14 Diet:LFD | Treatment:PFHxS RAW_FILE_NAME=rf14 SUBJECT_SAMPLE_FACTORS - S15 Diet:LFD | Treatment:PFHxS RAW_FILE_NAME=rf15 SUBJECT_SAMPLE_FACTORS - S16 Diet:LFD | Treatment:PFHxS RAW_FILE_NAME=rf16 SUBJECT_SAMPLE_FACTORS - S17 Diet:LFD | Treatment:PFHxS RAW_FILE_NAME=rf17 SUBJECT_SAMPLE_FACTORS - S18 Diet:LFD | Treatment:PFHxS RAW_FILE_NAME=rf18 SUBJECT_SAMPLE_FACTORS - S19 Diet:HFD | Treatment:none RAW_FILE_NAME=rf19 SUBJECT_SAMPLE_FACTORS - S20 Diet:HFD | Treatment:none RAW_FILE_NAME=rf20 SUBJECT_SAMPLE_FACTORS - S21 Diet:HFD | Treatment:none RAW_FILE_NAME=rf21 SUBJECT_SAMPLE_FACTORS - S22 Diet:HFD | Treatment:none RAW_FILE_NAME=rf22 SUBJECT_SAMPLE_FACTORS - S23 Diet:HFD | Treatment:none RAW_FILE_NAME=rf23 SUBJECT_SAMPLE_FACTORS - S24 Diet:HFD | Treatment:none RAW_FILE_NAME=rf24 SUBJECT_SAMPLE_FACTORS - S25 Diet:HFD | Treatment:PFOS RAW_FILE_NAME=rf25 SUBJECT_SAMPLE_FACTORS - S26 Diet:HFD | Treatment:PFOS RAW_FILE_NAME=rf26 SUBJECT_SAMPLE_FACTORS - S27 Diet:HFD | Treatment:PFOS RAW_FILE_NAME=rf27 SUBJECT_SAMPLE_FACTORS - S28 Diet:HFD | Treatment:PFOS RAW_FILE_NAME=rf28 SUBJECT_SAMPLE_FACTORS - S29 Diet:HFD | Treatment:PFOS RAW_FILE_NAME=rf29 SUBJECT_SAMPLE_FACTORS - S30 Diet:HFD | Treatment:PFOS RAW_FILE_NAME=rf30 SUBJECT_SAMPLE_FACTORS - S31 Diet:HFD | Treatment:PFHxS RAW_FILE_NAME=rf31 SUBJECT_SAMPLE_FACTORS - S32 Diet:HFD | Treatment:PFHxS RAW_FILE_NAME=rf32 SUBJECT_SAMPLE_FACTORS - S33 Diet:HFD | Treatment:PFHxS RAW_FILE_NAME=rf33 SUBJECT_SAMPLE_FACTORS - S34 Diet:HFD | Treatment:PFHxS RAW_FILE_NAME=rf34 SUBJECT_SAMPLE_FACTORS - S35 Diet:HFD | Treatment:PFHxS RAW_FILE_NAME=rf35 SUBJECT_SAMPLE_FACTORS - S36 Diet:HFD | Treatment:PFHxS RAW_FILE_NAME=rf36 #COLLECTION CO:COLLECTION_SUMMARY The study investigated diet-PFAS interactions and the impact of CO:COLLECTION_SUMMARY perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic (PFHxS) on the CO:COLLECTION_SUMMARY hepatic proteome and blood lipidomic profiles. The results supported the CO:COLLECTION_SUMMARY hypothesis that PFOS and PFHxS increase the risk of metabolic and inflammatory CO:COLLECTION_SUMMARY disease induced by diet. CO:SAMPLE_TYPE Blood (whole) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The mice were fed either a 10.5% kcal, low fat diet (LFD) (D12328, Research TR:TREATMENT_SUMMARY Diets, New Brunswick), or a 58% kcal, high fat diet (HFD) (D12331, Research TR:TREATMENT_SUMMARY Diets, New Brunswick). The mice were assigned to either diet alone, as controls, TR:TREATMENT_SUMMARY or to diet containing 0.0003% PFOS or 0.0003% PFHxS. The resulting treatment TR:TREATMENT_SUMMARY groups were as follows: low fat diet (LFD), high fat high carbohydrate diet TR:TREATMENT_SUMMARY (HFHC), LFD + PFOS (LPFNA), HFHC + PFOS (HPFOS), LFD + PFHxS (LPFHxS), and HFHC TR:TREATMENT_SUMMARY + PFHxS (HPFHxS) at n = 6 per treatment group. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Blood lipids were isolated for lipidomic analysis according to the Bligh and SP:SAMPLEPREP_SUMMARY Dyer method (Bligh and Dyer 1959). The lipidomics was performed at the SP:SAMPLEPREP_SUMMARY University of Georgia (Athens, GA). Briefly, blood samples designated for SP:SAMPLEPREP_SUMMARY lipidomics were suspended in 1.25 ml of methanol and 1.25 ml of chloroform. SP:SAMPLEPREP_SUMMARY Tubes were vortexed for 30 s, allowed to sit for 10 min on ice, centrifuged (300 SP:SAMPLEPREP_SUMMARY x g; 5 min), and the bottom chloroform layer was transferred to a new test tube. SP:SAMPLEPREP_SUMMARY The extraction steps were repeated three times and the chloroform layer SP:SAMPLEPREP_SUMMARY combined. A commercial mix of SPLASH Lipidomix internal standards (Avanti Polar SP:SAMPLEPREP_SUMMARY Lipids, Inc.) were spiked into each sample. SPLASH Lipidomix Mass Spec standards SP:SAMPLEPREP_SUMMARY includes all major lipid classes at ratios similar to that found in human SP:SAMPLEPREP_SUMMARY plasma. The collected chloroform layers were dried under nitrogen, reconstituted SP:SAMPLEPREP_SUMMARY with 50 µl of methanol: chloroform (3:1 v/v), and stored at 80ºC until SP:SAMPLEPREP_SUMMARY analysis. Lipid content was quantified by determining the level of inorganic SP:SAMPLEPREP_SUMMARY phosphorus using the Bartlett Assay (Bartlett 1959). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo-Fisher LTQ Orbitrap Elite CH:COLUMN_NAME Bruker Micron Magic nanoC18 (130mm X 100 µm, 5 µm) CH:FLOW_RATE 450-500 nL/min CH:SOLVENT_A 0.1% formic acid/water CH:SOLVENT_B mobile phase B was 0.1% formic acid/acetonitrile CH:INJECTION_TEMPERATURE 7 °C CH:TIME_PROGRAM 60 mins CH:TARGET_SAMPLE_TEMPERATURE 7 °C CH:CHROMATOGRAPHY_COMMENTS nanoC18 column (length, 130 mm; i.d., 100 μm; particle size, 5 μm; pore size, CH:CHROMATOGRAPHY_COMMENTS 150 Å; max flow rate, 500 nL/min; packing material, Bruker Micron Magic 18) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Lipid structures were identified based on the retention time and subsequent MS:MS_COMMENTS MS/MS spectra. Essentially, we determined structural information through MS:MS_COMMENTS LC-MS/MS and normalization of available lipid standards using LipidMatch. First, MS:MS_COMMENTS lipidomics data processed lipid features using MZmine as described in (Koelmel MS:MS_COMMENTS et al. 2017). Features observed in the blanks were removed using the blank MS:MS_COMMENTS feature filtration method (Patterson et al. 2017). The blank feature filtration MS:MS_COMMENTS method compared to various other filtering methods has been shown to increase MS:MS_COMMENTS the removal of true negatives while decreasing the removal of true positives MS:MS_COMMENTS (Patterson et al. 2017). The resulting MZmine features were annotated using MS:MS_COMMENTS LipidMatch (Koelmel et al. 2017). These annotations are putative, as annotations MS:MS_COMMENTS are based on in-silico MS/MS spectral libraries without matching internal MS:MS_COMMENTS standards for validation and without confirmation using orthogonal approaches MS:MS_COMMENTS (Sumner et al. 2007). The lipid match program then provided a single point MS:MS_COMMENTS calibration using exogenous lipid internal calibrant that best represents the MS:MS_COMMENTS lipid feature (based on lipid class, adduct and retention time). An R script was MS:MS_COMMENTS applied that combined multiple lipid features (adducts) into one feature 4 MS:MS_COMMENTS representing a unique lipid molecule. All open source lipidomics tools are MS:MS_COMMENTS published and available at http://secim.ufl.edu/ secim-tools/. MS:MS_RESULTS_FILE ST001525_AN002546_Results.txt UNITS:unknown Has m/z:Yes Has RT:Yes RT units:Minutes #END