#METABOLOMICS WORKBENCH kcbauer_20200424_143020_mwtab.txt DATATRACK_ID:1992 STUDY_ID:ST001366 ANALYSIS_ID:AN002274 PROJECT_ID:PR000934 VERSION 1 CREATED_ON April 27, 2020, 10:54 am #PROJECT PR:PROJECT_TITLE Malnutrition, Microbes, and Hippocampal Metabolomics PR:PROJECT_SUMMARY Examining the role of malnutrition and gut microbes on CNS function using the PR:PROJECT_SUMMARY MAL-BG murine model. RP-UPLC-FTMS was conducted on murine hippocampal tissue. PR:INSTITUTE University of British Columbia PR:DEPARTMENT Microbiology and Immunology PR:LABORATORY Finlay PR:LAST_NAME Bauer PR:FIRST_NAME Kylynda PR:ADDRESS #301 – 2185 East Mall, University of British Columbia Vancouver, British PR:ADDRESS Columbia Canada, V6T 1Z4 PR:EMAIL kcbauer@msl.ubc.ca PR:PHONE (604) 822-2210 PR:CONTRIBUTORS B. Brett Finlay #STUDY ST:STUDY_TITLE Malnutrition and Liver Metabolomics ST:STUDY_SUMMARY RP-UPLC-FTMS (+/- ion detection) were conducted on hippocampi from healthy (CON) ST:STUDY_SUMMARY and malnourished (MAL) mice, and MAL-BG (malnutrition plus E.coli/Bacteroidales ST:STUDY_SUMMARY exposure) mice. ST:INSTITUTE University of British Columbia ST:DEPARTMENT Microbiology and Immunology ST:LABORATORY B. Brett Finlay ST:LAST_NAME Bauer ST:FIRST_NAME Kylynda ST:ADDRESS Michael Smith Laboratories, #301 – 2185 East Mall, University of British ST:ADDRESS Columbia Vancouver, British Columbia Canada, V6T 1Z4 ST:EMAIL kcbauer@msl.ubc.ca ST:PHONE (604) 822-2210 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 15 ST:NUM_FEMALES 15 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 7 weeks SU:GENDER Female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratories #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 - CON_1 Treatment:CON | Diet:Healthy | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - CON_2 Treatment:CON | Diet:Healthy | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - CON_3 Treatment:CON | Diet:Healthy | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - CON_4 Treatment:CON | Diet:Healthy | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - CON_5 Treatment:CON | Diet:Healthy | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - MAL_1 Treatment:MAL | Diet:Malnourished | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - MAL_2 Treatment:MAL | Diet:Malnourished | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - MAL_3 Treatment:MAL | Diet:Malnourished | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - MAL_4 Treatment:MAL | Diet:Malnourished | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - MAL_5 Treatment:MAL | Diet:Malnourished | Bacterial Exposure:No SUBJECT_SAMPLE_FACTORS - MALBG_1 Treatment:MAL-BG | Diet:Malnourished | Bacterial Exposure:Yes SUBJECT_SAMPLE_FACTORS - MALBG_2 Treatment:MAL-BG | Diet:Malnourished | Bacterial Exposure:Yes SUBJECT_SAMPLE_FACTORS - MALBG_3 Treatment:MAL-BG | Diet:Malnourished | Bacterial Exposure:Yes SUBJECT_SAMPLE_FACTORS - MALBG_4 Treatment:MAL-BG | Diet:Malnourished | Bacterial Exposure:Yes SUBJECT_SAMPLE_FACTORS - MALBG_5 Treatment:MAL-BG | Diet:Malnourished | Bacterial Exposure:Yes #COLLECTION CO:COLLECTION_SUMMARY Following sacrifice, murine liver lobes were isolated and stored at -70/80 C CO:COLLECTION_SUMMARY prior to untargeted metabolomics. CO:SAMPLE_TYPE Hippocampus #TREATMENT TR:TREATMENT_SUMMARY CON = control mice on healthy diet, MAL = mice placed on a protein/fat deficient TR:TREATMENT_SUMMARY diet (malnourished), MAL-BG = MAL mice plus iterative E.coli/Bacteroidales TR:TREATMENT_SUMMARY exposure #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Methods reported in methodology .zip folder / Metabolomics were completed by SP:SAMPLEPREP_SUMMARY TMIC (The Metabolomics Innovation Centre). Each mouse hippocampal sample in an SP:SAMPLEPREP_SUMMARY Eppendorf tube was mixed with water, 5 μL per mg of the tissue, and two 4-mm SP:SAMPLEPREP_SUMMARY metal balls were added. The tissue was homogenized on a MM 400 mill mixer at a SP:SAMPLEPREP_SUMMARY vibrating frequency of 30 Hz for 1 min twice. After 5-s spin-down, a mixture of SP:SAMPLEPREP_SUMMARY methanol-chloroform (4:1) was added, at 25 μL per mg tissue, to each tube. The SP:SAMPLEPREP_SUMMARY sample was homogenized again for metabolite extraction using the same setup for SP:SAMPLEPREP_SUMMARY 1 min twice, followed by sonication in an ice-water bath for 5 min. The tube was SP:SAMPLEPREP_SUMMARY centrifuged at 15,000 rpm and at 10 0C for 20 min. The clear supernatant was SP:SAMPLEPREP_SUMMARY transferred to a 1.5-mL Eppendorf tube. A 60-μL aliquot from each sample was SP:SAMPLEPREP_SUMMARY dried down inside the same nitrogen evaporator and the residue was reconstituted SP:SAMPLEPREP_SUMMARY in 40 μL of 80% methanol. 10 μL was injected for reversed-phase SP:SAMPLEPREP_SUMMARY (RP)-UPLC-FTMS. Two rounds of sample injections were made, with positive- and SP:SAMPLEPREP_SUMMARY negative-ion detection, respectively. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters BEH C8 (2.1 x 50 mm, 1.7 µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo LTQ-Orbitrap Velos Pro MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS For relative quantitation, the MS instrument was run in the survey scan mode MS:MS_COMMENTS with FTMS detection at a mass resolution of 60,000 FWHM at m/z 400. The mass MS:MS_COMMENTS scan range was m/z 80 to 1800, with a reference lock-mass for real-time MS:MS_COMMENTS calibration. Two UPLC-FTMS datasets were acquired for each sample, one with MS:MS_COMMENTS positive-ion detection and the other with negative-ion detection. LC-MS/MS data MS:MS_COMMENTS was also acquired from each sample set with collision induced dissociation (CID) MS:MS_COMMENTS at different levels of normalized collision energy (from publication methods). MS:MS_RESULTS_FILE ST001366_AN002274_Results.txt UNITS:m/z values Has m/z:Yes Has RT:Yes RT units:Minutes #END