#METABOLOMICS WORKBENCH cnoecker_20230322_170413 DATATRACK_ID:3810 STUDY_ID:ST002518 ANALYSIS_ID:AN004147 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Systems biology illuminates the alternative metabolic niche of the human gut PR:PROJECT_TITLE bacterium Eggerthella lenta PR:PROJECT_TYPE Untargeted LC-MS PR:PROJECT_SUMMARY Human gut bacteria perform diverse metabolic functions with consequences for PR:PROJECT_SUMMARY host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta PR:PROJECT_SUMMARY performs several unusual chemical transformations, but it does not metabolize PR:PROJECT_SUMMARY sugars and its core growth strategy remains unclear. To obtain a comprehensive PR:PROJECT_SUMMARY view of the metabolic network of E. lenta, we generated several complementary PR:PROJECT_SUMMARY resources: defined culture media, metabolomics profiles of strain isolates, and PR:PROJECT_SUMMARY a curated genome-scale metabolic reconstruction. Stable isotope-resolved PR:PROJECT_SUMMARY metabolomics revealed that E. lenta uses acetate as a key carbon source while PR:PROJECT_SUMMARY catabolizing arginine to generate ATP, traits which could be recapitulated in PR:PROJECT_SUMMARY silico by our updated metabolic model. We compared these in vitro findings with PR:PROJECT_SUMMARY metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying PR:PROJECT_SUMMARY shared signatures across environments and highlighting catabolism of the host PR:PROJECT_SUMMARY signaling metabolite agmatine as an alternative energy pathway. Together, our PR:PROJECT_SUMMARY results elucidate a distinctive metabolic niche filled by E. lenta in the gut PR:PROJECT_SUMMARY ecosystem. PR:INSTITUTE University of California, San Francisco PR:DEPARTMENT Microbiology and Immunology PR:LABORATORY Peter Turnbaugh PR:LAST_NAME Noecker PR:FIRST_NAME Cecilia PR:ADDRESS 513 Parnassus Ave HSW1501, San Francisco, CA 94143 PR:EMAIL cecilia.noecker@ucsf.edu PR:PHONE 415-502-3264 PR:FUNDING_SOURCE This work was supported by the National Institutes of Health (2R01HL122593; PR:FUNDING_SOURCE 1R01AT011117; 1R01DK114034 to P.J.T., F32GM140808 to C.N.). P.J.T. is a Chan PR:FUNDING_SOURCE Zuckerberg Biohub Investigator and held an Investigators in the Pathogenesis of PR:FUNDING_SOURCE Infectious Disease Award from the Burroughs Wellcome Fund. PR:PUBLICATIONS https://doi.org/10.1101/2022.09.19.508335 PR:DOI http://dx.doi.org/10.21228/M89B04 #STUDY ST:STUDY_TITLE Time course 3: Growth of Eggerthella lenta in defined media with some samples ST:STUDY_TITLE receiving 13C6 stable isotope labeled arginine ST:STUDY_TYPE Untargeted LC-MS ST:STUDY_SUMMARY This dataset contains untargeted metabolomics analysis of supernatants from ST:STUDY_SUMMARY Eggerthella lenta DSM 2243 grown in defined EDM1 media. One set of samples grew ST:STUDY_SUMMARY in EDM1 containing 13C6 stable isotope labeled arginine. ST:INSTITUTE University of California, San Francisco ST:LAST_NAME Noecker ST:FIRST_NAME Cecilia ST:ADDRESS 513 Parnassus Ave HSW1501, San Francisco, CA 94143 ST:EMAIL cecilia.noecker@ucsf.edu ST:PHONE 415-502-3264 ST:SUBMIT_DATE 2023-03-22 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Eggerthella lenta SU:TAXONOMY_ID 84112 SU:GENOTYPE_STRAIN DSM 2243 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - MSA0029 Strain:2243 | ArgGroup:HR | TimePoint:TP0 | Time:0 Sample=HR_2243_TP0_2; F_ID=F29; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0029.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0029.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0029.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0029.mzML SUBJECT_SAMPLE_FACTORS - MSA0043 Strain:2243 | ArgGroup:HR | TimePoint:TP0 | Time:0 Sample=HR_2243_TP0_1; F_ID=F43; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0043.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0043.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0043.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0043.mzML SUBJECT_SAMPLE_FACTORS - MSA0050 Strain:2243 | ArgGroup:HR | TimePoint:TP0 | Time:0 Sample=HR_2243_TP0_3; F_ID=F50; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0050.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0050.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0050.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0050.mzML SUBJECT_SAMPLE_FACTORS - MSA0004 Strain:2243 | ArgGroup:HR | TimePoint:TP1 | Time:20 Sample=HR_2243_TP1_3; F_ID=F4; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0004.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0004.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0004.mzML SUBJECT_SAMPLE_FACTORS - MSA0049 Strain:2243 | ArgGroup:HR | TimePoint:TP1 | Time:20 Sample=HR_2243_TP1_1; F_ID=F49; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0049.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0049.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0049.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0049.mzML SUBJECT_SAMPLE_FACTORS - MSA0056 Strain:2243 | ArgGroup:HR | TimePoint:TP1 | Time:20 Sample=HR_2243_TP1_2; F_ID=F56; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0056.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0056.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0056.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0056.mzML SUBJECT_SAMPLE_FACTORS - MSA0031 Strain:2243 | ArgGroup:HR | TimePoint:TP2 | Time:32 Sample=HR_2243_TP2_1; F_ID=F31; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0031.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0031.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0031.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0031.mzML SUBJECT_SAMPLE_FACTORS - MSA0032 Strain:2243 | ArgGroup:HR | TimePoint:TP2 | Time:32 Sample=HR_2243_TP2_3; F_ID=F32; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0032.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0032.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0032.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0032.mzML SUBJECT_SAMPLE_FACTORS - MSA0064 Strain:2243 | ArgGroup:HR | TimePoint:TP2 | Time:32 Sample=HR_2243_TP2_2; F_ID=F64; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0064.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0064.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0064.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0064.mzML SUBJECT_SAMPLE_FACTORS - MSA0007 Strain:2243 | ArgGroup:HR | TimePoint:TP3 | Time:44 Sample=HR_2243_TP3_1; F_ID=F7; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0007.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0007.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0007.mzML SUBJECT_SAMPLE_FACTORS - MSA0014 Strain:2243 | ArgGroup:HR | TimePoint:TP3 | Time:44 Sample=HR_2243_TP3_2; F_ID=F14; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0014.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0014.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0014.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0014.mzML SUBJECT_SAMPLE_FACTORS - MSA0025 Strain:2243 | ArgGroup:HR | TimePoint:TP3 | Time:44 Sample=HR_2243_TP3_3; F_ID=F25; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0025.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0025.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0025.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0025.mzML SUBJECT_SAMPLE_FACTORS - MSA0044 Strain:2243 | ArgGroup:HR | TimePoint:TP4 | Time:56 Sample=HR_2243_TP4_3; F_ID=F44; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0044.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0044.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0044.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0044.mzML SUBJECT_SAMPLE_FACTORS - MSA0053 Strain:2243 | ArgGroup:HR | TimePoint:TP4 | Time:56 Sample=HR_2243_TP4_2; F_ID=F53; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0053.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0053.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0053.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0053.mzML SUBJECT_SAMPLE_FACTORS - MSA0060 Strain:2243 | ArgGroup:HR | TimePoint:TP4 | Time:56 Sample=HR_2243_TP4_1; F_ID=F60; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0060.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0060.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0060.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0060.mzML SUBJECT_SAMPLE_FACTORS - MSA0013 Strain:2243 | ArgGroup:R | TimePoint:TP0 | Time:0 Sample=R_2243_TP0_1; F_ID=F13; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0013.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0013.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0013.mzML SUBJECT_SAMPLE_FACTORS - MSA0054 Strain:2243 | ArgGroup:R | TimePoint:TP0 | Time:0 Sample=R_2243_TP0_3; F_ID=F54; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0054.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0054.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0054.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0054.mzML SUBJECT_SAMPLE_FACTORS - MSA0055 Strain:2243 | ArgGroup:R | TimePoint:TP0 | Time:0 Sample=R_2243_TP0_2; F_ID=F55; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0055.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0055.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0055.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0055.mzML SUBJECT_SAMPLE_FACTORS - MSA0018 Strain:2243 | ArgGroup:R | TimePoint:TP1 | Time:20 Sample=R_2243_TP1_3; F_ID=F18; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0018.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0018.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0018.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0018.mzML SUBJECT_SAMPLE_FACTORS - MSA0033 Strain:2243 | ArgGroup:R | TimePoint:TP1 | Time:20 Sample=R_2243_TP1_2; F_ID=F33; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0033.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0033.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0033.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0033.mzML SUBJECT_SAMPLE_FACTORS - MSA0048 Strain:2243 | ArgGroup:R | TimePoint:TP1 | Time:20 Sample=R_2243_TP1_1; F_ID=F48; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0048.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0048.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0048.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0048.mzML SUBJECT_SAMPLE_FACTORS - MSA0017 Strain:2243 | ArgGroup:R | TimePoint:TP2 | Time:32 Sample=R_2243_TP2_2; F_ID=F17; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0017.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0017.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0017.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0017.mzML SUBJECT_SAMPLE_FACTORS - MSA0026 Strain:2243 | ArgGroup:R | TimePoint:TP2 | Time:32 Sample=R_2243_TP2_3; F_ID=F26; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0026.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0026.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0026.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0026.mzML SUBJECT_SAMPLE_FACTORS - MSA0042 Strain:2243 | ArgGroup:R | TimePoint:TP2 | Time:32 Sample=R_2243_TP2_1; F_ID=F42; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0042.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0042.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0042.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0042.mzML SUBJECT_SAMPLE_FACTORS - MSA0008 Strain:2243 | ArgGroup:R | TimePoint:TP3 | Time:44 Sample=R_2243_TP3_1; F_ID=F8; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0008.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0008.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0008.mzML SUBJECT_SAMPLE_FACTORS - MSA0009 Strain:2243 | ArgGroup:R | TimePoint:TP3 | Time:44 Sample=R_2243_TP3_3; F_ID=F9; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0009.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0009.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0009.mzML SUBJECT_SAMPLE_FACTORS - MSA0039 Strain:2243 | ArgGroup:R | TimePoint:TP3 | Time:44 Sample=R_2243_TP3_2; F_ID=F39; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0039.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0039.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0039.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0039.mzML SUBJECT_SAMPLE_FACTORS - MSA0001 Strain:2243 | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_2243_TP4_2; F_ID=F1; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0001.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0001.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0001.mzML SUBJECT_SAMPLE_FACTORS - MSA0002 Strain:2243 | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_2243_TP4_1; F_ID=F2; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0002.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0002.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0002.mzML SUBJECT_SAMPLE_FACTORS - MSA0034 Strain:2243 | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_2243_TP4_3; F_ID=F34; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0034.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0034.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0034.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0034.mzML SUBJECT_SAMPLE_FACTORS - BK1 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_BK1.raw; RAW_FILE_NAME=M00182_20220405Pos_BK1.raw; RAW_FILE_NAME=M00182_20220405Neg_BK1.mzML; RAW_FILE_NAME=M00182_20220405Pos_BK1.mzML SUBJECT_SAMPLE_FACTORS - BK2 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_BK2.raw; RAW_FILE_NAME=M00182_20220405Pos_BK2.raw; RAW_FILE_NAME=M00182_20220405Neg_BK2.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - BK3 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_BK3.raw; RAW_FILE_NAME=M00182_20220405Pos_BK3.raw; RAW_FILE_NAME=M00182_20220405Neg_BK3.mzML; RAW_FILE_NAME=M00182_20220405Pos_BK3.mzML SUBJECT_SAMPLE_FACTORS - BK4 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_BK4.raw; RAW_FILE_NAME=M00182_20220405Pos_BK4.raw; RAW_FILE_NAME=M00182_20220405Neg_BK4.mzML; RAW_FILE_NAME=M00182_20220405Pos_BK4.mzML SUBJECT_SAMPLE_FACTORS - BK5 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_BK5.raw; RAW_FILE_NAME=M00182_20220405Pos_BK5.raw; RAW_FILE_NAME=M00182_20220405Neg_BK5.mzML; RAW_FILE_NAME=M00182_20220405Pos_BK5.mzML SUBJECT_SAMPLE_FACTORS - BK6 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_BK6.raw; RAW_FILE_NAME=M00182_20220405Pos_BK6.raw; RAW_FILE_NAME=M00182_20220405Neg_BK6.mzML; RAW_FILE_NAME=M00182_20220405Pos_BK6.mzML SUBJECT_SAMPLE_FACTORS - BK7 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_BK7.raw; RAW_FILE_NAME=M00182_20220405Pos_BK7.raw; RAW_FILE_NAME=M00182_20220405Neg_BK7.mzML; RAW_FILE_NAME=M00182_20220405Pos_BK7.mzML SUBJECT_SAMPLE_FACTORS - QC1 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_QC1.raw; RAW_FILE_NAME=M00182_20220405Pos_QC1.raw; RAW_FILE_NAME=M00182_20220405Neg_QC1.mzML; RAW_FILE_NAME=M00182_20220405Pos_QC1.mzML SUBJECT_SAMPLE_FACTORS - QC2 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_QC2.raw; RAW_FILE_NAME=M00182_20220405Pos_QC2.raw; RAW_FILE_NAME=M00182_20220405Neg_QC2.mzML; RAW_FILE_NAME=M00182_20220405Pos_QC2.mzML SUBJECT_SAMPLE_FACTORS - QC3 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_QC3.raw; RAW_FILE_NAME=M00182_20220405Pos_QC3.raw; RAW_FILE_NAME=M00182_20220405Neg_QC3.mzML; RAW_FILE_NAME=M00182_20220405Pos_QC3.mzML SUBJECT_SAMPLE_FACTORS - QC4 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_QC4.raw; RAW_FILE_NAME=M00182_20220405Pos_QC4.raw; RAW_FILE_NAME=M00182_20220405Neg_QC4.mzML; RAW_FILE_NAME=M00182_20220405Pos_QC4.mzML SUBJECT_SAMPLE_FACTORS - QC5 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_QC5.raw; RAW_FILE_NAME=M00182_20220405Pos_QC5.raw; RAW_FILE_NAME=M00182_20220405Neg_QC5.mzML; RAW_FILE_NAME=M00182_20220405Pos_QC5.mzML SUBJECT_SAMPLE_FACTORS - QC6 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_QC6.raw; RAW_FILE_NAME=M00182_20220405Pos_QC6.raw; RAW_FILE_NAME=M00182_20220405Neg_QC6.mzML; RAW_FILE_NAME=M00182_20220405Pos_QC6.mzML SUBJECT_SAMPLE_FACTORS - QC7 Strain:- | ArgGroup:- | TimePoint:- | Time:- Sample=-; F_ID=-; RAW_FILE_NAME=M00182_20220405Neg_QC7.raw; RAW_FILE_NAME=M00182_20220405Pos_QC7.raw; RAW_FILE_NAME=M00182_20220405Neg_QC7.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - MSA0028 Strain:c | ArgGroup:HR | TimePoint:TP0 | Time:0 Sample=HR_c_TP0_2; F_ID=F28; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0028.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0028.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0028.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0028.mzML SUBJECT_SAMPLE_FACTORS - MSA0059 Strain:c | ArgGroup:HR | TimePoint:TP0 | Time:0 Sample=HR_c_TP0_3; F_ID=F59; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0059.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0059.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0059.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0059.mzML SUBJECT_SAMPLE_FACTORS - MSA0062 Strain:c | ArgGroup:HR | TimePoint:TP0 | Time:0 Sample=HR_c_TP0_1; F_ID=F62; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0062.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0062.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0062.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0062.mzML SUBJECT_SAMPLE_FACTORS - MSA0012 Strain:c | ArgGroup:HR | TimePoint:TP1 | Time:20 Sample=HR_c_TP1_3; F_ID=F12; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0012.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0012.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0012.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0012.mzML SUBJECT_SAMPLE_FACTORS - MSA0024 Strain:c | ArgGroup:HR | TimePoint:TP1 | Time:20 Sample=HR_c_TP1_1; F_ID=F24; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0024.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0024.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0024.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0024.mzML SUBJECT_SAMPLE_FACTORS - MSA0046 Strain:c | ArgGroup:HR | TimePoint:TP1 | Time:20 Sample=HR_c_TP1_2; F_ID=F46; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0046.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0046.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0046.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0046.mzML SUBJECT_SAMPLE_FACTORS - MSA0030 Strain:c | ArgGroup:HR | TimePoint:TP2 | Time:32 Sample=HR_c_TP2_2; F_ID=F30; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0030.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0030.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0030.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0030.mzML SUBJECT_SAMPLE_FACTORS - MSA0038 Strain:c | ArgGroup:HR | TimePoint:TP2 | Time:32 Sample=HR_c_TP2_3; F_ID=F38; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0038.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0038.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0038.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0038.mzML SUBJECT_SAMPLE_FACTORS - MSA0057 Strain:c | ArgGroup:HR | TimePoint:TP2 | Time:32 Sample=HR_c_TP2_1; F_ID=F57; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0057.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0057.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0057.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0057.mzML SUBJECT_SAMPLE_FACTORS - MSA0016 Strain:c | ArgGroup:HR | TimePoint:TP3 | Time:44 Sample=HR_c_TP3_1; F_ID=F16; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0016.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0016.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0016.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0016.mzML SUBJECT_SAMPLE_FACTORS - MSA0027 Strain:c | ArgGroup:HR | TimePoint:TP3 | Time:44 Sample=HR_c_TP3_3; F_ID=F27; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0027.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0027.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0027.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - MSA0035 Strain:c | ArgGroup:HR | TimePoint:TP3 | Time:44 Sample=HR_c_TP3_2; F_ID=F35; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0035.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0035.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0035.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0035.mzML SUBJECT_SAMPLE_FACTORS - MSA0015 Strain:c | ArgGroup:HR | TimePoint:TP4 | Time:56 Sample=HR_c_TP4_3; F_ID=F15; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0015.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0015.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0015.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0015.mzML SUBJECT_SAMPLE_FACTORS - MSA0036 Strain:c | ArgGroup:HR | TimePoint:TP4 | Time:56 Sample=HR_c_TP4_1; F_ID=F36; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0036.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0036.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0036.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0036.mzML SUBJECT_SAMPLE_FACTORS - MSA0047 Strain:c | ArgGroup:HR | TimePoint:TP4 | Time:56 Sample=HR_c_TP4_2; F_ID=F47; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0047.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0047.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0047.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0047.mzML SUBJECT_SAMPLE_FACTORS - MSA0045 Strain:c | ArgGroup:R | TimePoint:TP0 | Time:0 Sample=R_c_TP0_3; F_ID=F45; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0045.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0045.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0045.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - MSA0052 Strain:c | ArgGroup:R | TimePoint:TP0 | Time:0 Sample=R_c_TP0_2; F_ID=F52; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0052.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0052.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0052.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0052.mzML SUBJECT_SAMPLE_FACTORS - MSA0061 Strain:c | ArgGroup:R | TimePoint:TP0 | Time:0 Sample=R_c_TP0_1; F_ID=F61; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0061.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0061.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0061.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0061.mzML SUBJECT_SAMPLE_FACTORS - MSA0010 Strain:c | ArgGroup:R | TimePoint:TP1 | Time:20 Sample=R_c_TP1_1; F_ID=F10; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0010.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0010.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0010.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0010.mzML SUBJECT_SAMPLE_FACTORS - MSA0021 Strain:c | ArgGroup:R | TimePoint:TP1 | Time:20 Sample=R_c_TP1_3; F_ID=F21; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0021.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0021.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0021.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0021.mzML SUBJECT_SAMPLE_FACTORS - MSA0041 Strain:c | ArgGroup:R | TimePoint:TP1 | Time:20 Sample=R_c_TP1_2; F_ID=F41; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0041.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0041.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0041.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0041.mzML SUBJECT_SAMPLE_FACTORS - MSA0019 Strain:c | ArgGroup:R | TimePoint:TP2 | Time:32 Sample=R_c_TP2_3; F_ID=F19; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0019.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0019.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0019.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0019.mzML SUBJECT_SAMPLE_FACTORS - MSA0022 Strain:c | ArgGroup:R | TimePoint:TP2 | Time:32 Sample=R_c_TP2_2; F_ID=F22; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0022.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0022.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0022.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0022.mzML SUBJECT_SAMPLE_FACTORS - MSA0023 Strain:c | ArgGroup:R | TimePoint:TP2 | Time:32 Sample=R_c_TP2_1; F_ID=F23; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0023.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0023.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0023.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0023.mzML SUBJECT_SAMPLE_FACTORS - MSA0006 Strain:c | ArgGroup:R | TimePoint:TP3 | Time:44 Sample=R_c_TP3_1; F_ID=F6; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0006.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0006.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0006.mzML SUBJECT_SAMPLE_FACTORS - MSA0020 Strain:c | ArgGroup:R | TimePoint:TP3 | Time:44 Sample=R_c_TP3_3; F_ID=F20; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0020.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0020.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0020.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0020.mzML SUBJECT_SAMPLE_FACTORS - MSA0058 Strain:c | ArgGroup:R | TimePoint:TP3 | Time:44 Sample=R_c_TP3_2; F_ID=F58; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0058.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0058.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0058.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0058.mzML SUBJECT_SAMPLE_FACTORS - MSA0003 Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_c_TP4_2_extra4; F_ID=F3; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0003.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0003.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0003.mzML SUBJECT_SAMPLE_FACTORS - MSA0005 Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_c_TP4_1; F_ID=F5; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0005.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0005.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0005.mzML SUBJECT_SAMPLE_FACTORS - MSA0011 Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_c_TP4_2_extra1; F_ID=F11; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0011.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0011.raw; RAW_FILE_NAME=-; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0011.mzML SUBJECT_SAMPLE_FACTORS - MSA0037 Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_c_TP4_2_extra2; F_ID=F37; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0037.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0037.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0037.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0037.mzML SUBJECT_SAMPLE_FACTORS - MSA0040 Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_c_TP4_2_extra3; F_ID=F40; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0040.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0040.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0040.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0040.mzML SUBJECT_SAMPLE_FACTORS - MSA0051 Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_c_TP4_2; F_ID=F51; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0051.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0051.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0051.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0051.mzML SUBJECT_SAMPLE_FACTORS - MSA0063 Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56 Sample=R_c_TP4_3; F_ID=F63; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0063.raw; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0063.raw; RAW_FILE_NAME=M00182_20220405_Neg_1_MSA0063.mzML; RAW_FILE_NAME=M00182_20220405_Pos_1_MSA0063.mzML #COLLECTION CO:COLLECTION_SUMMARY Time course experiments were conducted in tubes in the anaerobic chamber in a CO:COLLECTION_SUMMARY 37°C incubator. For all metabolomics experiments, three independent culture CO:COLLECTION_SUMMARY replicates were included for each condition, with an equal number of CO:COLLECTION_SUMMARY uninoculated control tubes. Starter cultures and inocula were prepared as CO:COLLECTION_SUMMARY described above for growth assays. 5mLs of defined media was added to VWR glass CO:COLLECTION_SUMMARY culture tubes (53283-800) with screw caps. The PBS-washed inoculum was added to CO:COLLECTION_SUMMARY culture tubes to obtain an approximate starting OD600 of 0.001. A preliminary CO:COLLECTION_SUMMARY growth assay was conducted to define time points spanning the exponential growth CO:COLLECTION_SUMMARY phase in the tested conditions. At each time point, OD600 measurements of all CO:COLLECTION_SUMMARY inoculated tubes were first measured using a Hach DR1900 spectrophotometer, with CO:COLLECTION_SUMMARY a paired control tube to normalize for the background. 100 μL from each tube CO:COLLECTION_SUMMARY were then transferred into a 96-well microplate, which was sealed and removed CO:COLLECTION_SUMMARY from the anaerobic chamber. Plates were centrifuged at 1,928 rcf at 4°C for 8 CO:COLLECTION_SUMMARY minutes, after which supernatants were collected into fresh polypropylene tubes CO:COLLECTION_SUMMARY or plates, sealed, and flash-frozen in liquid nitrogen. Two time course CO:COLLECTION_SUMMARY experiments were carried out with stable isotope-labeled substrates. CO:COLLECTION_SUMMARY Experimental groups included conditions in which sodium arginine in the defined CO:COLLECTION_SUMMARY media was replaced with 13C6 labeled arginine HCl, along with a matched CO:COLLECTION_SUMMARY experimental group with the same concentration of unlabeled substrate. CO:SAMPLE_TYPE Bacterial culture supernatant CO:COLLECTION_FREQUENCY at time points specified in study design table over 56 hours (full growth phase) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For growth and metabolomics experiments, glycerol stocks of the 3 E. lenta TR:TREATMENT_SUMMARY strains were first streaked on BHI+ agar plates and incubated at 37°C for 2-3 TR:TREATMENT_SUMMARY days. Individual colonies were inoculated into 3-4 mL liquid BHI+ and incubated TR:TREATMENT_SUMMARY at 37°C for 40-48 hours, or until approximately early stationary phase. Culture TR:TREATMENT_SUMMARY optical density (600 nm wavelength absorbance, OD600) was measured using a Hach TR:TREATMENT_SUMMARY DR1900 spectrophotometer. 1 mL samples of BHI starter cultures were then TR:TREATMENT_SUMMARY centrifuged at 1,568 rcf for 4 minutes in a microcentrifuge (ThermoScientific TR:TREATMENT_SUMMARY mySpin 12) in the anaerobic chamber and resuspended in 1 mL sterile TR:TREATMENT_SUMMARY phosphate-buffered saline (PBS). The resulting suspension was vortexed and TR:TREATMENT_SUMMARY diluted to an approximate OD600 of 0.1, and used as inoculum into defined TR:TREATMENT_SUMMARY experimental conditions. Varying media conditions were prepared separately and TR:TREATMENT_SUMMARY all allowed to fully reduce in the anaerobic chamber prior to inoculation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Bacterial culture supernatant and sterile media, used in culture, were thawed on SP:SAMPLEPREP_SUMMARY wet ice. Once thawed, samples were homogenized by inversion five times. SP:SAMPLEPREP_SUMMARY Extracellular culture supernatant samples were prepared as follows: 20 μL of SP:SAMPLEPREP_SUMMARY culture supernatant were extracted using 80 μL of a chilled extraction solvent SP:SAMPLEPREP_SUMMARY at −20°C (1:1 acetonitrile:methanol, 5% water containing stable SP:SAMPLEPREP_SUMMARY isotope-labeled internal standards). Samples were homogenized via pipette SP:SAMPLEPREP_SUMMARY action, incubated for 1 hour at −20°C, centrifuged at 4°C at 6000 rcf for 5 SP:SAMPLEPREP_SUMMARY min. The supernatant was transferred to a new plate and immediately sealed and SP:SAMPLEPREP_SUMMARY kept at 4°C prior to prompt analysis via LC-MS/MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples, sterile media, pools, and blanks were promptly added to a Thermo CH:CHROMATOGRAPHY_SUMMARY Vanquish Autosampler at 4°C in a Vanquish UHPLC (Thermo Fisher Scientific, CH:CHROMATOGRAPHY_SUMMARY Waltham, MA). Chromatographic separation was performed using an ACQUITY Bridged CH:CHROMATOGRAPHY_SUMMARY Ethylene Hybrid (BEH) Amide column 2.1 x 150 mm, 1.7-micron particle size, CH:CHROMATOGRAPHY_SUMMARY (Waters Corp. Milford, MA), using chromatographic conditions published elsewhere CH:CHROMATOGRAPHY_SUMMARY (HILIC method described in the Supplementary Methods of CH:CHROMATOGRAPHY_SUMMARY doi.org/10.1038/s41586-021-03707-9). CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) CH:COLUMN_TEMPERATURE 40 CH:FLOW_GRADIENT The gradient profile was held at 100% B for 2 minutes, from 100% B to 70% B in 5 CH:FLOW_GRADIENT minutes, holding at 70% B for 0.7 minute, from 70% B to 40% B for 1.3 minutes, CH:FLOW_GRADIENT holding at 40% B for 0.5 minutes, from 40% B to 30% B for 0.75 minutes, before CH:FLOW_GRADIENT returning to 100% B for 2.5 minutes and holding at 100% B for 4 minutes. CH:FLOW_RATE 400 μL per minute CH:SOLVENT_A 100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3 CH:SOLVENT_B 95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 MS:MS_COMMENTS selection of metabolites from our in-house ‘local’ library, when additional MS:MS_COMMENTS scan bandwidth was available MS2 was collected in a data-dependent manner. Mass MS:MS_COMMENTS range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was MS:MS_COMMENTS collected, loop count was 4, isolation window was 1.5 Da. In SIRM samples, MS:MS_COMMENTS deuterated internal standards were replaced with CUDA and Val-Tyr-Val to enable MS:MS_COMMENTS untargeted enrichment analysis. LC-MS/MS analysis conditions for SIRM MS:MS_COMMENTS metabolomics were identical to those used for standard untargeted metabolomics. MS:MS_COMMENTS Intra- and extracellular untargeted data generated from SIRM experiments was MS:MS_COMMENTS analyzed separately using Compound Discoverer version 3.3 (Thermo Scientific, MS:MS_COMMENTS Bremen, Germany). Samples treated with labeled compounds were always paired with MS:MS_COMMENTS matched samples treated with unlabeled compounds in order to correct for MS:MS_COMMENTS naturally occurring isotope abundances. Unlabeled samples were used for compound MS:MS_COMMENTS detection and formula assignment via isotope pattern-based prediction, spectral MS:MS_COMMENTS library matches, or mass lists matches. The isotope patterns and formulas from MS:MS_COMMENTS the sample files then served as a reference for the detection of potential MS:MS_COMMENTS isotopologues per compound in the labeled sample type. A specification of the MS:MS_COMMENTS full Compound Discoverer workflow is available at MS:MS_COMMENTS https://github.com/turnbaughlab/2022_Noecker_ElentaMetabolism. MS:ION_MODE POSITIVE #END