#METABOLOMICS WORKBENCH Codreags00_20230615_084710 DATATRACK_ID:4092 STUDY_ID:ST002737 ANALYSIS_ID:AN004439 PROJECT_ID:PR001702 VERSION 1 CREATED_ON June 16, 2023, 8:24 am #PROJECT PR:PROJECT_TITLE 2’-fucosyllactose modulates gut microbial metabolism for the prevention of PR:PROJECT_TITLE colitis PR:PROJECT_TYPE Untargeted Metabolomics analysis PR:PROJECT_SUMMARY Human milk-derived 2’-fucosyllactose (2’-FL) consumption is associated with PR:PROJECT_SUMMARY health benefits in infancy that extend into adulthood. However, the exact PR:PROJECT_SUMMARY biological functions of 2’-FL and corresponding mechanisms of action remain PR:PROJECT_SUMMARY largely unknown. Here, we investigated the impact of 2’-FL on gut microbial PR:PROJECT_SUMMARY metabolism for the prevention of colitis in adulthood. The gut microbiota from PR:PROJECT_SUMMARY adult mice treated with 2’-FL showed an increase in abundance of several PR:PROJECT_SUMMARY health-associated genera, including Bifidobacterium, and exhibited preventive PR:PROJECT_SUMMARY effects on colitis. Microbial metabolic analysis demonstrated that 26 pathways PR:PROJECT_SUMMARY that are significantly different between non-inflammatory bowel disease PR:PROJECT_SUMMARY individuals and patients with ulcerative colitis (UC) are significantly PR:PROJECT_SUMMARY regulated by 2’-FL in mice, indicating that 2’-FL has the potential to PR:PROJECT_SUMMARY directly regulate dysregulated microbial metabolism in UC. Exploratory PR:PROJECT_SUMMARY metabolomics of Bifidobacterium infantis identified novel secreted metabolites PR:PROJECT_SUMMARY significantly enriched by 2’-FL consumption, including pantothenol. PR:PROJECT_SUMMARY Remarkably, pantothenate significantly protects mucosal barrier and mitigates PR:PROJECT_SUMMARY colitis in adult mice. Thus 2’-FL-modulated gut microbial metabolism may PR:PROJECT_SUMMARY contribute to the prevention of intestinal inflammation in adulthood. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME CODREANU PR:FIRST_NAME SIMONA PR:ADDRESS 1234 STEVENSON CENTER LANE PR:EMAIL SIMONA.CODREANU@VANDERBILT.EDU PR:PHONE 6158758422 #STUDY ST:STUDY_TITLE 2’-fucosyllactose prevents colitis ST:STUDY_TYPE untargeted metabolomics analysis ST:STUDY_SUMMARY Human milk-derived 2’-fucosyllactose (2’-FL) consumption is associated with ST:STUDY_SUMMARY health benefits in infancy that extend into adulthood. However, the exact ST:STUDY_SUMMARY biological functions of 2’-FL and corresponding mechanisms of action remain ST:STUDY_SUMMARY largely unknown. Here, we investigated the impact of 2’-FL on gut microbial ST:STUDY_SUMMARY metabolism for the prevention of colitis in adulthood. The gut microbiota from ST:STUDY_SUMMARY adult mice treated with 2’-FL showed an increase in abundance of several ST:STUDY_SUMMARY health-associated genera, including Bifidobacterium, and exhibited preventive ST:STUDY_SUMMARY effects on colitis. Microbial metabolic analysis demonstrated that 26 pathways ST:STUDY_SUMMARY that are significantly different between non-inflammatory bowel disease ST:STUDY_SUMMARY individuals and patients with ulcerative colitis (UC) are significantly ST:STUDY_SUMMARY regulated by 2’-FL in mice, indicating that 2’-FL has the potential to ST:STUDY_SUMMARY directly regulate dysregulated microbial metabolism in UC. Exploratory ST:STUDY_SUMMARY metabolomics of Bifidobacterium infantis identified novel secreted metabolites ST:STUDY_SUMMARY significantly enriched by 2’-FL consumption, including pantothenol. ST:STUDY_SUMMARY Remarkably, pantothenate significantly protects mucosal barrier and mitigates ST:STUDY_SUMMARY colitis in adult mice. Thus 2’-FL-modulated gut microbial metabolism may ST:STUDY_SUMMARY contribute to the prevention of intestinal inflammation in adulthood. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME CODREANU ST:FIRST_NAME SIMONA ST:ADDRESS 1234 STEVENSON CENTER LANE ST:EMAIL SIMONA.CODREANU@VANDERBILT.EDU ST:PHONE 6158758422 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 10 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Bifidobacterium longum subspecies infantis (B. infantis) SU:GENOTYPE_STRAIN ATCC 15702 and ATCC 15697 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC #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 Control-1 C_1 treatment:0 RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_C01 SUBJECT_SAMPLE_FACTORS Control-2 C_2 treatment:0 RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_C02 SUBJECT_SAMPLE_FACTORS Control-3 C_3 treatment:0 RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_C03 SUBJECT_SAMPLE_FACTORS Control-4 C_4 treatment:0 RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_C04 SUBJECT_SAMPLE_FACTORS Control-5 C_5 treatment:0 RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_C05 SUBJECT_SAMPLE_FACTORS 2FL-1 F_1 treatment:2'FL RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_F01 SUBJECT_SAMPLE_FACTORS 2FL-2 F_2 treatment:2'FL RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_F02 SUBJECT_SAMPLE_FACTORS 2FL-3 F_3 treatment:2'FL RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_F03 SUBJECT_SAMPLE_FACTORS 2FL-4 F_4 treatment:2'FL RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_F04 SUBJECT_SAMPLE_FACTORS 2FL-5 F_5 treatment:2'FL RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_F05 SUBJECT_SAMPLE_FACTORS Media only MC_01 treatment:0 RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_MC01 SUBJECT_SAMPLE_FACTORS Media only MC_02 treatment:0 RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_MC02 SUBJECT_SAMPLE_FACTORS Media only MC_03 treatment:0 RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_MC03 SUBJECT_SAMPLE_FACTORS Media_2FL MF_01 treatment:2'FL RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_MF01 SUBJECT_SAMPLE_FACTORS Media_2FL MF_02 treatment:2'FL RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_MF02 SUBJECT_SAMPLE_FACTORS Media_2FL MF_03 treatment:2'FL RAW_FILE_NAME=SC_20211207_RPLCp_FMS_Yan_MF03 #COLLECTION CO:COLLECTION_SUMMARY B. infantis 15702 were inoculated in RCM at 37C with or without CO:COLLECTION_SUMMARY supplementation with 10 mg/mL of 2’-FL (1.0% w/v) until OD600≈1. Bacteria CO:COLLECTION_SUMMARY were pelleted and supernatants were filtered (0.2 micron) and stored at -80ºC CO:COLLECTION_SUMMARY for untargeted metabolomics. Cultured RCM medium and 10 mg/mL 2’-FL CO:COLLECTION_SUMMARY supplemented RCM medium without bacteria inoculation served as controls. CO:COLLECTION_SUMMARY Experiments were repeated 2-3 times for collecting five biological replicates CO:COLLECTION_SUMMARY from each group. CO:SAMPLE_TYPE Bacterial cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY B. infantis 15702 were inoculated in RCM at 37C with or without TR:TREATMENT_SUMMARY supplementation with 10 mg/mL of 2’-FL (1.0% w/v) until OD600≈1. Bacteria TR:TREATMENT_SUMMARY were pelleted and supernatants were filtered (0.2 micron) and stored at -80ºC TR:TREATMENT_SUMMARY for untargeted metabolomics. Cultured RCM medium and 10 mg/mL 2’-FL TR:TREATMENT_SUMMARY supplemented RCM medium without bacteria inoculation served as controls. TR:TREATMENT_SUMMARY Experiments were repeated 2-3 times for collecting five biological replicates TR:TREATMENT_SUMMARY from each group. TR:TREATMENT 2’-fucosyllactose TR:TREATMENT_DOSE 10mg/mL #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Bifidobacteria infantis were cultured in Reinforced Clostridial Medium (RCM) SP:SAMPLEPREP_SUMMARY with or without 2’-fucosyllactose (2’-FL), a human milk oligosaccharide. SP:SAMPLEPREP_SUMMARY Bacteria were pelleted by centrifugation and supernatants were then collected, SP:SAMPLEPREP_SUMMARY snap frozen, and stored at -80°C until analyzed via Liquid Chromatography-High SP:SAMPLEPREP_SUMMARY Resolution Mass Spectrometry (LC-HRMS and LC-HRMS/MS)-based metabolomics in the SP:SAMPLEPREP_SUMMARY Vanderbilt Center for Innovative Technology using previously described methods. SP:SAMPLEPREP_SUMMARY Briefly, equal volumes (200µL) of previously frozen culture medium were SP:SAMPLEPREP_SUMMARY prepared. Isotopically labeled standards, biotin-D2 and phenylalanine-D8, were SP:SAMPLEPREP_SUMMARY added to individual samples to assess the sample preparation steps. Samples were SP:SAMPLEPREP_SUMMARY subjected to protein precipitation by addition of 800µL of ice-cold methanol, SP:SAMPLEPREP_SUMMARY then incubated at -80C overnight. Following protein precipitation, samples SP:SAMPLEPREP_SUMMARY were centrifuged at 10,000 rpm for 10 min to remove insoluble material. SP:SAMPLEPREP_SUMMARY Supernatant(s) were transferred and dried in vacuo, and stored at -80C prior SP:SAMPLEPREP_SUMMARY to MS characterization. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Following lysis and standard addition, protein precipitation was performed by SP:EXTRACTION_METHOD adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at SP:EXTRACTION_METHOD -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm SP:EXTRACTION_METHOD for 10 min to eliminate proteins. The supernatants containing metabolites were SP:EXTRACTION_METHOD dried via speed-vacuum. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For the RPLC analysis metabolite extracts (5μL injection volume) were separated CH:CHROMATOGRAPHY_SUMMARY on a Hypersil Gold, 1.9 µm, 2.1mm x 100 mm column (Thermo Fisher) held at CH:CHROMATOGRAPHY_SUMMARY 40°C. Liquid chromatography was performed at a 250μL min-1 using solvent A CH:CHROMATOGRAPHY_SUMMARY (0.1% formic acid (FA) in water) and solvent B (0.1% FA in acetonitrile:water CH:CHROMATOGRAPHY_SUMMARY 80:20) over a 30 min gradient. Mass spectrometry analyses were performed in CH:CHROMATOGRAPHY_SUMMARY positive ion mode with the parameters as previously published except for the CH:CHROMATOGRAPHY_SUMMARY following changes. First, tandem mass spectra were acquired using a data CH:CHROMATOGRAPHY_SUMMARY dependent scanning mode in which one full MS scan (m/z 70-1050) was followed by CH:CHROMATOGRAPHY_SUMMARY 2 or 10 MS/MS scans. MS/MS scans are acquired in profile mode using an isolation CH:CHROMATOGRAPHY_SUMMARY width of 1.3 m/z, stepped collision energy (NCE 20, 40), and a dynamic exclusion CH:CHROMATOGRAPHY_SUMMARY of 6 s. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Vanquish UHPLC binary system CH:COLUMN_NAME Thermo Hypersil Gold (100 x 2.1mm, 1.9um) CH:SOLVENT_A 100% water, 0.1% Formic Acid CH:SOLVENT_B 80:20 acetonitrile:water, 0.1% Formic Acid CH:FLOW_GRADIENT 30 min CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The acquired RPLC-HRMS raw data from five (5) biological replicates from each MS:MS_COMMENTS sample type were imported, processed, normalized and reviewed using Progenesis MS:MS_COMMENTS QI v.3.0 (Non-linear Dynamics, Newcastle, UK). All MS and MS/MS sample runs were MS:MS_COMMENTS aligned against a pooled QC reference run. Unique ions (retention time and m/z MS:MS_COMMENTS pairs) were de-adducted and de-isotoped to generate unique “features” (or MS:MS_COMMENTS retention time and m/z pairs). Data were normalized to all features and cleaned MS:MS_COMMENTS by removing spectral features >25% CV in the pooled QC samples. Sample process MS:MS_COMMENTS and instrument variability were also assessed to determine sample acceptance. MS:MS_COMMENTS Briefly, QA metrics for sample process variability and instrument variability MS:MS_COMMENTS are ≤10% CV and ≤5% CV, respectively. In these studies, no samples were MS:MS_COMMENTS identified as outliers. Statistical analyses were performed in Progensis QI MS:MS_COMMENTS using variance stabilized measurements achieved through log normalization to MS:MS_COMMENTS calculate p-values by one-way analysis of variance (ANOVA) test. Significantly MS:MS_COMMENTS changed metabolites were chosen with the criteria p-value <0.05 and |FC| > 2. MS:MS_RESULTS_FILE ST002737_AN004439_Results.txt UNITS:time_m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END