#METABOLOMICS WORKBENCH hwelhaven_20221019_123521 DATATRACK_ID:3522 STUDY_ID:ST002342 ANALYSIS_ID:AN003826 VERSION 1 CREATED_ON 12-15-2022 #PROJECT PR:PROJECT_TITLE Welhaven_Vahidi_JBMR PR:PROJECT_SUMMARY C57BL/6J male and female germ-free and conventional mice were housed at Montana PR:PROJECT_SUMMARY State University and then used to study differences in bone associated with PR:PROJECT_SUMMARY microbiome status and sex. PR:INSTITUTE Montana State University PR:LAST_NAME Welhaven PR:FIRST_NAME Hope PR:ADDRESS Culbertson Hall, 100 PR:EMAIL hwelhaven@gmail.com PR:PHONE 4066961526 PR:FUNDING_SOURCE National Science Foundation (CMMI 1554708, CMMI 2120239) and the National PR:FUNDING_SOURCE Institutes of Health (NIAMS R01AR073964, NIGMS P20GM103474, NIH R03AG068680 PR:DOI http://dx.doi.org/10.21228/M88X2F #STUDY ST:STUDY_TITLE The gut microbiome has sexually dimorphic effects on bone tissue energy ST:STUDY_TITLE metabolism and multiscale bone quality in C57BL/6J mice ST:STUDY_SUMMARY Male and female germ-free and conventional were euthanized at 20 weeks of age, ST:STUDY_SUMMARY and various tissues were obtained to assess differences in bone properties ST:STUDY_SUMMARY associated with microbiome status and sex. For metabolomic analyses, the humeri ST:STUDY_SUMMARY was obtained, bone marrow was flushed using PBS, and subchondral bone ST:STUDY_SUMMARY metabolites were extracted using a methanol:acetone precipitation approach. ST:STUDY_SUMMARY Next, samples were injected, ionized, and data was acquired using LC-MS. Data ST:STUDY_SUMMARY was processed and converted using MSConvert and XCMS. Following this step, ST:STUDY_SUMMARY metabolic phenotypes of mice that differ by microbiome status and sex were ST:STUDY_SUMMARY investigated using MetaboAnalyst. Differences in metabolism were related to ST:STUDY_SUMMARY lipid, energy, and amino acid metabolism. Specifically, females, both ST:STUDY_SUMMARY conventional and germ-free, had dysregulated lipid metabolism, whereas, males, ST:STUDY_SUMMARY both conventional and germ-free had dysregulated amino acid metabolism. Other ST:STUDY_SUMMARY differences detected between male and female mice that differ by germ-free ST:STUDY_SUMMARY status corresponded to differences in bone strength, bone marrow adiposity, and ST:STUDY_SUMMARY osteocyte density. ST:INSTITUTE Montana State University ST:DEPARTMENT Mechanical & Industrial Engineering, Chemistry & Biochemistry ST:LAST_NAME Welhaven ST:FIRST_NAME Hope ST:ADDRESS Culbertson Hall, 100 ST:EMAIL hwelhaven@gmail.com ST:PHONE 4066961526 ST:SUBMIT_DATE 2022-10-19 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57Bl/6 SU:AGE_OR_AGE_RANGE 20 weeks (at euthanasia) SU:GENDER Male and female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labratories SU:ANIMAL_LIGHT_CYCLE Standard light dark cycle SU:ANIMAL_FEED PicoLab Rodent Diet 20, 20% protein #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - H27 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H27 SUBJECT_SAMPLE_FACTORS - H28 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H28 SUBJECT_SAMPLE_FACTORS - H29 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H29 SUBJECT_SAMPLE_FACTORS - H30 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H30 SUBJECT_SAMPLE_FACTORS - H31 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H31 SUBJECT_SAMPLE_FACTORS - H32 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H32 SUBJECT_SAMPLE_FACTORS - H33 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H33 SUBJECT_SAMPLE_FACTORS - H34 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H34 SUBJECT_SAMPLE_FACTORS - H35 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H35 SUBJECT_SAMPLE_FACTORS - H36 Sex:Female | Microbiome Status:Control RAW_FILE_NAME=H36 SUBJECT_SAMPLE_FACTORS - H1 Sex:Female | Microbiome Status:Germ-Free RAW_FILE_NAME=H1 SUBJECT_SAMPLE_FACTORS - H18 Sex:Female | Microbiome Status:Germ-Free RAW_FILE_NAME=H18 SUBJECT_SAMPLE_FACTORS - H19 Sex:Female | Microbiome Status:Germ-Free RAW_FILE_NAME=H19 SUBJECT_SAMPLE_FACTORS - H2 Sex:Female | Microbiome Status:Germ-Free RAW_FILE_NAME=H2 SUBJECT_SAMPLE_FACTORS - H20 Sex:Female | Microbiome Status:Germ-Free RAW_FILE_NAME=H20 SUBJECT_SAMPLE_FACTORS - H3 Sex:Female | Microbiome Status:Germ-Free RAW_FILE_NAME=H3 SUBJECT_SAMPLE_FACTORS - H10 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H10 SUBJECT_SAMPLE_FACTORS - H11 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H11 SUBJECT_SAMPLE_FACTORS - H12 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H12 SUBJECT_SAMPLE_FACTORS - H13 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H13 SUBJECT_SAMPLE_FACTORS - H4 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H4 SUBJECT_SAMPLE_FACTORS - H5 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H5 SUBJECT_SAMPLE_FACTORS - H6 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H6 SUBJECT_SAMPLE_FACTORS - H7 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H7 SUBJECT_SAMPLE_FACTORS - H8 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H8 SUBJECT_SAMPLE_FACTORS - H9 Sex:Male | Microbiome Status:Control RAW_FILE_NAME=H9 SUBJECT_SAMPLE_FACTORS - H14 Sex:Male | Microbiome Status:Germ-Free RAW_FILE_NAME=H14 SUBJECT_SAMPLE_FACTORS - H15 Sex:Male | Microbiome Status:Germ-Free RAW_FILE_NAME=H15 SUBJECT_SAMPLE_FACTORS - H16 Sex:Male | Microbiome Status:Germ-Free RAW_FILE_NAME=H16 SUBJECT_SAMPLE_FACTORS - H17 Sex:Male | Microbiome Status:Germ-Free RAW_FILE_NAME=H17 SUBJECT_SAMPLE_FACTORS - H21 Sex:Male | Microbiome Status:Germ-Free RAW_FILE_NAME=H21 SUBJECT_SAMPLE_FACTORS - H22 Sex:Male | Microbiome Status:Germ-Free RAW_FILE_NAME=H22 SUBJECT_SAMPLE_FACTORS - H23 Sex:Male | Microbiome Status:Germ-Free RAW_FILE_NAME=H23 #COLLECTION CO:COLLECTION_SUMMARY Samples were collected following mouse euthanasia. The humerus was obtained and CO:COLLECTION_SUMMARY bone marrow was flushed using PBS. Next, metabolites were extracted using CO:COLLECTION_SUMMARY methanol:acetone. CO:SAMPLE_TYPE Bone #TREATMENT TR:TREATMENT_SUMMARY All C57Bl/6J mice (male, female, germ-free, conventional) were housed at Montana TR:TREATMENT_SUMMARY State University. During this time, mice were kept on a light/dark cycle and fed TR:TREATMENT_SUMMARY a standard chow diet ad libitum. Upon 20 weeks of age, mice were euthanized. No TR:TREATMENT_SUMMARY additional factors such as injury, diet, or exercise occurred. Mice were TR:TREATMENT_SUMMARY employed to analyze differences in bone at macro and micro levels that are TR:TREATMENT_SUMMARY associated with the microbiome and sex. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Once euthanized, tissues were dissected and harvested for various techniques. SP:SAMPLEPREP_SUMMARY For metabolomics, humeri samples underwent homogenization/crushing, extracted SP:SAMPLEPREP_SUMMARY with methanol:acetone, and were then subjected to rounds of vortexing and SP:SAMPLEPREP_SUMMARY centrifugation. Once metabolites were isolated, samples were dried via vacuum SP:SAMPLEPREP_SUMMARY concentration and resuspended with acetonitrile:water for LC-MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed using an Agilent 1290 UPLC coupled to an Agilent 6538 CH:CHROMATOGRAPHY_SUMMARY QTOF. All samples were run in normal phase using a Cogent Diamond Hydride HILIC CH:CHROMATOGRAPHY_SUMMARY column (150 x 2.1 mm; 100Å pore size on a 4µm particle size). Solvents: 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid in water (A), 0.1% formic acid in acetonitrile (B). Elution CH:CHROMATOGRAPHY_SUMMARY gradient: 100 to 25% solvent B over 17 minutes, 0 to 70% solvent A over 20 CH:CHROMATOGRAPHY_SUMMARY minutes, at 21 minutes, B increases from 25% to 100% till 25 minutes while A CH:CHROMATOGRAPHY_SUMMARY decreases from 70% to 0% till 25 minutes. The last 4 minutes act as a wash. CH:CHROMATOGRAPHY_SUMMARY Total run time = 25 minute method (per sample) CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Cogent Diamond Hydride (150 × 2.1mm,4um) CH:FLOW_GRADIENT 100 to 25% solvent B over 17 minutes, 0 to 70% solvent A over 20 minutes, at 21 CH:FLOW_GRADIENT minutes, B increases from 25% to 100% till 25 minutes while A decreases from 70% CH:FLOW_GRADIENT to 0% till 25 minutes. The last 4 minutes act as a wash. CH:FLOW_RATE 0.400 mL/min CH:INJECTION_TEMPERATURE 4C CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:ANALYTICAL_TIME 25 minutes CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS AN:OPERATOR_NAME Hope Welhaven #MS MS:INSTRUMENT_NAME Agilent 6538 QTOF MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS Data was acquired in positive mode following a 25-minute method. Following MS:MS_COMMENTS injection and acquisition, .d files were converted to .mzMLs using MSConvert MS:MS_COMMENTS (Threshold peak filter value = 1000; Peak picking = true, 1-1; 32-bit, no z-lib MS:MS_COMMENTS compression). Once .mzMLs were avaiable, XCMS was used (parameters reflected run MS:MS_COMMENTS parameters = positive mode, feature detection 15 ppm, adducts = [M+H]+, [M+Na]+ MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002342_AN003826_Results.txt UNITS:Peak Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END