#METABOLOMICS WORKBENCH ericmichbrown_20250421_193510 DATATRACK_ID:5858 STUDY_ID:ST003941 ANALYSIS_ID:AN006477 PROJECT_ID:PR002470 VERSION 1 CREATED_ON June 2, 2025, 7:09 pm #PROJECT PR:PROJECT_TITLE Sphingolipids promote anti-inflammatory responses of Bacteroides through the PR:PROJECT_TITLE mevalonate pathway PR:PROJECT_TYPE Bacterial lipidomics PR:PROJECT_SUMMARY A lipidomics analysis to identify sphingolipids of interest in Bacteroides PR:PROJECT_SUMMARY thetaiotaomicron outer membrane vesicles (OMVs) and whole cell bacteria. PR:INSTITUTE Broad Institute of MIT and Harvard PR:DEPARTMENT IDMP PR:LABORATORY Xavier Lab and Clish Lab PR:LAST_NAME Brown PR:FIRST_NAME Eric PR:ADDRESS 415 Main Street, Cambridge, Massachusetts, 02142, USA PR:EMAIL erbrown@broadinstitute.org PR:PHONE 6176826566 PR:CONTRIBUTORS Clary Clish, Sarah Jeanfavre, Julian Avila-Pacheco #STUDY ST:STUDY_TITLE Lipidomic analysis of Bacteroides thetaiotaomicron WT vs SPT KO ST:STUDY_TITLE (sphingolipid-deficient) outer membrane vesicles (OMVs) and whole cells. ST:STUDY_SUMMARY We performed a lipidomic analysis of outer membrane vesicles (OMVs) from ST:STUDY_SUMMARY Bacteroides thetaiotaomicron vs whole cell bacteria to understand whether there ST:STUDY_SUMMARY are lipids enriched in the OMVs of Bacteroides. We also utilized a ST:STUDY_SUMMARY sphingolipid-deficient Bacteroides thetaiotaomicron strain with a knockout of ST:STUDY_SUMMARY the serine palmitoyltransferase gene (spt) and compared the lipidomic signature ST:STUDY_SUMMARY to the wild-type strain in OMVs and whole cell bacteria. Overall we report the ST:STUDY_SUMMARY differences in lipids from each strain in OMVs and in whole cell bacteria and ST:STUDY_SUMMARY which sphingolipids are enriched in OMVs. ST:INSTITUTE Broad Institute of MIT and Harvard ST:DEPARTMENT IDMP ST:LABORATORY Xavier Lab and Clish Lab ST:LAST_NAME Brown ST:FIRST_NAME Eric ST:ADDRESS 415 Main Street, Cambridge, MA, USA 02142 ST:EMAIL ericmichbrown@gmail.com ST:PHONE 6176826566 ST:STUDY_COMMENTS Bacterial lipidomics on OMVs and whole cell bacteria on B. thetaiotaomicron WT ST:STUDY_COMMENTS vs SPT KO strains #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Bacteroides thetaiotaomicron SU:TAXONOMY_ID 8188 SU:GENOTYPE_STRAIN Wild-type vs SPT Knockout strain #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 - BTWTOMV_1 Sample source:OMVs | Genotype:Wild-type | Sample type:OMVs RAW_FILE_NAME(Raw file name)=Sphingolipid-BTWT-OMV_1.raw SUBJECT_SAMPLE_FACTORS - BTWTOMV_2 Sample source:OMVs | Genotype:Wild-type | Sample type:OMVs RAW_FILE_NAME(Raw file name)=Sphingolipid-BTWT-OMV_2.raw SUBJECT_SAMPLE_FACTORS - BTWTOMV_3 Sample source:OMVs | Genotype:Wild-type | Sample type:OMVs RAW_FILE_NAME(Raw file name)=Sphingolipid-BTWT-OMV_3.raw SUBJECT_SAMPLE_FACTORS - BTSPTOMV_1 Sample source:OMVs | Genotype:SPT KO | Sample type:OMVs RAW_FILE_NAME(Raw file name)=Sphingolipid-BTSPT-OMV_1.raw SUBJECT_SAMPLE_FACTORS - BTSPTOMV_2 Sample source:OMVs | Genotype:SPT KO | Sample type:OMVs RAW_FILE_NAME(Raw file name)=Sphingolipid-BTSPT-OMV_2.raw SUBJECT_SAMPLE_FACTORS - BTSPTOMV_3 Sample source:OMVs | Genotype:SPT KO | Sample type:OMVs RAW_FILE_NAME(Raw file name)=Sphingolipid-BTSPT-OMV_3.raw SUBJECT_SAMPLE_FACTORS - BTWTWC_1 Sample source:Bacteria | Genotype:Wild-type | Sample type:Bacteria RAW_FILE_NAME(Raw file name)=Sphingolipid-BTWT-Cell-pellet_1.raw SUBJECT_SAMPLE_FACTORS - BTWTWC_2 Sample source:Bacteria | Genotype:Wild-type | Sample type:Bacteria RAW_FILE_NAME(Raw file name)=Sphingolipid-BTWT-Cell-pellet_2.raw SUBJECT_SAMPLE_FACTORS - BTWTWC_3 Sample source:Bacteria | Genotype:Wild-type | Sample type:Bacteria RAW_FILE_NAME(Raw file name)=Sphingolipid-BTWT-Cell-pellet_3.raw SUBJECT_SAMPLE_FACTORS - BTSPTWC_1 Sample source:Bacteria | Genotype:SPT KO | Sample type:Bacteria RAW_FILE_NAME(Raw file name)=Sphingolipid-BTSPT-Cell-pellet_1.raw SUBJECT_SAMPLE_FACTORS - BTSPTWC_2 Sample source:Bacteria | Genotype:SPT KO | Sample type:Bacteria RAW_FILE_NAME(Raw file name)=Sphingolipid-BTSPT-Cell-pellet_2.raw SUBJECT_SAMPLE_FACTORS - BTSPTWC_3 Sample source:Bacteria | Genotype:SPT KO | Sample type:Bacteria RAW_FILE_NAME(Raw file name)=Sphingolipid-BTSPT-Cell-pellet_3.raw #COLLECTION CO:COLLECTION_SUMMARY Bacteroides thetaiotaomicron wild-type or spt-knockout OMVs were collected after CO:COLLECTION_SUMMARY 2 days of culture in BHI media by ultra-centrifugation and frozen at -80 degrees CO:COLLECTION_SUMMARY until analysis. To obtain the whole cell lipidomic data, either wild-type or spt CO:COLLECTION_SUMMARY knockout Bacteroides thetaiotaomicron strains were centrifuged at 8000g for 20 CO:COLLECTION_SUMMARY min and pellets were snap frozen before analysis. CO:SAMPLE_TYPE Bacterial cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Cells were not treated TR:CELL_STORAGE -80 TR:CELL_MEDIA Brain Heart Infusion Agar #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were prepped by adding isopropanol to the OMVs or cells to extract SP:SAMPLEPREP_SUMMARY metabolites and then injected on the machine SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A - 95:5:0.1 vol/vol/vol 10 mM ammonium acetate/methanol/formic acid CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Waters ACQUITY BEH C8 (100 x 2.1mm, 1.7um) CH:SOLVENT_A 95% water/5% methanol; 10 mM ammonium acetate; 0.1% formic acid CH:SOLVENT_B 100% water; 0.1% formic acid CH:FLOW_GRADIENT The column was eluted isocratically with 80% A for 1 minute followed by a linear CH:FLOW_GRADIENT gradient to 80% B over 2 minutes, a linear gradient to 100% B over 7 minutes, CH:FLOW_GRADIENT then 3 minutes at 100% B CH:FLOW_RATE 0.45mL/min CH:COLUMN_TEMPERATURE 40 CH:METHODS_FILENAME Lipidomic_profiling_methods_Metabolomics_Workbench_Sphingolipids.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Lipidomic_profiling_methods_Metabolomics_Workbench_Sphingolipids.pdf #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS analyses were carried out using electrospray ionization in the positive ion MS:MS_COMMENTS mode using full scan analysis over 220–1500 m/z at 120,000 resolution. Other MS:MS_COMMENTS MS settings were: sheath gas 40, sweep gas 2, aux gas 10, spray voltage 3 kV, MS:MS_COMMENTS capillary temperature 290°C, RF lens 60, vaporizer temperature 325°C, MS:MS_COMMENTS microscans 1, and maximum injection time 100 ms. Raw data were processed using MS:MS_COMMENTS TraceFinder software (Thermo Fisher Scientific) for targeted peak integration MS:MS_COMMENTS and manual review of a subset of identified lipids and using Progenesis QI MS:MS_COMMENTS (Nonlinear Dynamics) for peak detection and integration of both annotated and MS:MS_COMMENTS unannotated lipids. Lipid identities were determined based on comparison to MS:MS_COMMENTS reference plasma extracts and are denoted by total number of carbons in the MS:MS_COMMENTS lipid acyl chain(s) and total number of double bonds in the lipid acyl chain(s). MS:MS_COMMENTS This was done in accordance with standard practices outlined by the Data MS:MS_COMMENTS Standards and Metabolite Identification Task Groups of the International MS:MS_COMMENTS Metabolomics Society. MS:MS_RESULTS_FILE ST003941_AN006477_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END