#METABOLOMICS WORKBENCH mososa4177_20201211_104021 DATATRACK_ID:2349 STUDY_ID:ST001651 ANALYSIS_ID:AN002697 PROJECT_ID:PR001057 VERSION 1 CREATED_ON January 13, 2021, 1:27 pm #PROJECT PR:PROJECT_TITLE Untargeted stable-isotope probing of the gut microbiota metabolome using PR:PROJECT_TITLE 13C-labeled dietary fibers PR:PROJECT_SUMMARY The gut microbiome generates numerous metabolites that exert local effects and PR:PROJECT_SUMMARY enter the circulation to affect the functions of many organs. Despite extensive PR:PROJECT_SUMMARY sequencing-based characterization of the gut microbiome, there remains a lack of PR:PROJECT_SUMMARY understanding of microbial metabolism. Here, we developed an untargeted stable PR:PROJECT_SUMMARY isotope-resolved metabolomics (SIRM) approach for the holistic study of gut PR:PROJECT_SUMMARY microbial metabolites. PR:INSTITUTE University of Kentucky PR:LAST_NAME Deng PR:FIRST_NAME Pan PR:ADDRESS 789 South Limestone PR:EMAIL pde233@uky.edu PR:PHONE 8595623039 #STUDY ST:STUDY_TITLE Analysis of metabolites in gut microbioal culture media and microbial cells ST:STUDY_SUMMARY We developed an untargeted stable isotope-resolved metabolomics (SIRM) approach ST:STUDY_SUMMARY for the holistic study of gut microbial metabolites ST:INSTITUTE University of Kentucky ST:LAST_NAME Deng ST:FIRST_NAME Pan ST:ADDRESS 789 South Limestone ST:EMAIL pde233@uky.edu ST:PHONE 8595623039 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Female #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 - Cellulose_Media_1 Treatment:13C-Cellulose Matrix=Media; RAW_FILE_NAME=20200312_POS_LFD_1_cellulose SUBJECT_SAMPLE_FACTORS - Cellulose_Media_2 Treatment:13C-Cellulose Matrix=Media; RAW_FILE_NAME=20200312_POS_LFD_2_cellulose SUBJECT_SAMPLE_FACTORS - Cellulose_Media_3 Treatment:13C-Cellulose Matrix=Media; RAW_FILE_NAME=20200312_POS_LFD_3_cellulose SUBJECT_SAMPLE_FACTORS - Inulin_Media_1 Treatment:13C-Inulin Matrix=Media; RAW_FILE_NAME=20200312_POS_LFD_1_inulin SUBJECT_SAMPLE_FACTORS - Inulin_Media_2 Treatment:13C-Inulin Matrix=Media; RAW_FILE_NAME=20200312_POS_LFD_2_inulin SUBJECT_SAMPLE_FACTORS - Inulin_Media_3 Treatment:13C-Inulin Matrix=Media; RAW_FILE_NAME=20200312_POS_LFD_3_inulin SUBJECT_SAMPLE_FACTORS - Cellulose_Cell_1 Treatment:13C-Cellulose Matrix=Cell; RAW_FILE_NAME=20200313_POS_LFD_1_cellulose SUBJECT_SAMPLE_FACTORS - Cellulose_Cell_2 Treatment:13C-Cellulose Matrix=Cell; RAW_FILE_NAME=20200313_POS_LFD_2_cellulose SUBJECT_SAMPLE_FACTORS - Cellulose_Cell_3 Treatment:13C-Cellulose Matrix=Cell; RAW_FILE_NAME=20200313_POS_LFD_3_cellulose SUBJECT_SAMPLE_FACTORS - Inulin_Cell_1 Treatment:13C-Inulin Matrix=Cell; RAW_FILE_NAME=20200313_POS_LFD_1_inulin SUBJECT_SAMPLE_FACTORS - Inulin_Cell_2 Treatment:13C-Inulin Matrix=Cell; RAW_FILE_NAME=20200313_POS_LFD_2_inulin SUBJECT_SAMPLE_FACTORS - Inulin_Cell_3 Treatment:13C-Inulin Matrix=Cell; RAW_FILE_NAME=20200313_POS_LFD_3_inulin #COLLECTION CO:COLLECTION_SUMMARY Fresh fecal pellets were collected from each mouse (23 weeks old) and CO:COLLECTION_SUMMARY immediately placed in a sterile microcentrifuge tube. Samples were quickly CO:COLLECTION_SUMMARY transferred to an anaerobic chamber for immediate experimental setup to ensure CO:COLLECTION_SUMMARY microbial viability. CO:SAMPLE_TYPE Feces CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY Fresh fecal samples (ca. 110 mg) from each mouse were weighed and dispensed in 3 TR:TREATMENT_SUMMARY mL of prepared medium separately in an anaerobic chamber. The samples were TR:TREATMENT_SUMMARY pestled to suspend the microorganisms and particles. The suspensions were TR:TREATMENT_SUMMARY subjected to low-speed centrifugation and the supernatants were then collected TR:TREATMENT_SUMMARY and centrifuged to pellet microbes. The microbial cells were then suspended in 4 TR:TREATMENT_SUMMARY mL of culture medium, and divided equally into two Hungate tubes. To each paired TR:TREATMENT_SUMMARY tube was amended with either 13C-Inulin or 13C-cellulose aseptically to achieve TR:TREATMENT_SUMMARY a final concentration of 4 g/L fibers. The sealed Hungate tubes were incubated TR:TREATMENT_SUMMARY in a water bath (37°C) for 24 h. After incubation, the samples were centrifuged TR:TREATMENT_SUMMARY (3,000 g, 5 min) to collect supernatant (culture medium). Each pellet was washed TR:TREATMENT_SUMMARY with 1 mL of fresh culture medium and centrifuged to collect the microbial TR:TREATMENT_SUMMARY cells. All procedures were performed under anaerobic conditions. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The collected medium was weighed, and equal amounts of medium (50% of total SP:SAMPLEPREP_SUMMARY medium weight) were accurately aliquoted from each sample and lyophilized. The SP:SAMPLEPREP_SUMMARY microbial cells were quenched using 450 μL cold methanol right after SP:SAMPLEPREP_SUMMARY collection. After brief agitation by vortex, the samples were transferred into SP:SAMPLEPREP_SUMMARY glass tubes. Then 5 mL of methyl tert-butyl ether was added to each tube and the SP:SAMPLEPREP_SUMMARY samples were mixed on a multi-tube vortexer (600 rpm, 30 min). Phase separation SP:SAMPLEPREP_SUMMARY was then induced by adding 1.25 mL of Millipore deionized water. Samples were SP:SAMPLEPREP_SUMMARY then vortexed again for 1 min, incubated at 4 °C for 10 min to allow phase SP:SAMPLEPREP_SUMMARY separation and centrifuged (3,000 g, 10 min, 4 °C). Polar fractions were SP:SAMPLEPREP_SUMMARY collected into clean tubes and lyophilized. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC-HILIC (150 x 2.1mm, 5um) CH:FLOW_GRADIENT 0–20 min, linear gradient from 80% to 20% B; 20–21 min, hold at 20% B min; CH:FLOW_GRADIENT 21-22 min, linear gradient to 80% B; 22-28 min, re-equilibrate at 80% B CH:FLOW_RATE 0.150 ml/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A 20 mM ammonium carbonate, 0.1% ammonium hydroxide CH:SOLVENT_B acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometer was operated in positive and negative ionization modes. MS:MS_COMMENTS Initial metabolite identification was performed using Compound Discoverer 3.1 MS:MS_COMMENTS (Thermo Fisher Scientific).Metabolites were further confirmed by comparison of MS:MS_COMMENTS the ion features in the samples with an in-house reference library of authentic MS:MS_COMMENTS chemical standards. Peak areas of metabolites and isotopologues were integrated MS:MS_COMMENTS and exported to Excel via the TraceFinder 5.0 software package (Thermo Fisher MS:MS_COMMENTS Scientific). MS:MS_RESULTS_FILE ST001651_AN002697_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END