#METABOLOMICS WORKBENCH schrodm_20230112_103651 DATATRACK_ID:3688 STUDY_ID:ST002441 ANALYSIS_ID:AN003978 PROJECT_ID:PR001572 VERSION 1 CREATED_ON January 13, 2023, 7:27 am #PROJECT PR:PROJECT_TITLE Impacts of interactions between environmental chemical exposures and diet on gut PR:PROJECT_TITLE microbiota and microbiota-derived metabolites in mothers and children PR:PROJECT_TYPE C18 Reversed-Phase Broad Spectrum Metabolomics PR:PROJECT_SUMMARY This project is funded by the NIH Environmental influences on Child Health PR:PROJECT_SUMMARY Outcomes (ECHO) OIF program, and uses data from two cohorts: MAAP and MARCH. PR:PROJECT_SUMMARY This project will examine associations with PFAS exposures and gut microbiota PR:PROJECT_SUMMARY form and function in mothers and children over time. Untargeted metabolomics PR:PROJECT_SUMMARY will be used as a means to assess changes in microbiota-derived metabolites. PR:INSTITUTE NC HHEAR Hub PR:DEPARTMENT Untargeted Analysis PR:LABORATORY Sumner Lab PR:LAST_NAME Li PR:FIRST_NAME Yuanyuan PR:ADDRESS Nutrition Research Institute, UNC-CH, 500 Laureate Way, Kannapolis, NC 28081 PR:EMAIL yuanyli4@unc.edu PR:PHONE 984-377-0693 PR:FUNDING_SOURCE This research was supported by the Environmental influences on Child Health PR:FUNDING_SOURCE Outcomes (ECHO) OIF program, Office of The Director, National Institutes of PR:FUNDING_SOURCE Health. The content is solely the responsibility of the authors and does not PR:FUNDING_SOURCE necessarily represent the official views of the National Institutes of Health. PR:FUNDING_SOURCE MARCH is an ECHO cohort which is supported by the following ECHO Program PR:FUNDING_SOURCE Collaborators: ECHO Coordinating Center: Duke Clinical Research Institute, PR:FUNDING_SOURCE Durham, North Carolina: Smith PB, Newby KL, Benjamin DK; U2C OD023375; ECHO Data PR:FUNDING_SOURCE Analysis Center: Johns Hopkins University Bloomberg School of Public Health, PR:FUNDING_SOURCE Baltimore, Maryland: Jacobson LP; Research Triangle Institute, Durham, North PR:FUNDING_SOURCE Carolina: Catellier.D U24 OD023382; North Carolina Human Health Exposure PR:FUNDING_SOURCE Analysis Resource Hub: Research Triangle Institute: Fennell T, University of PR:FUNDING_SOURCE North Carolina at Chapel Hill: Sumner S, University of North Carolina at PR:FUNDING_SOURCE Charlotte: Du X; U2C ES030857; Human Health Exposure Analysis Resource PR:FUNDING_SOURCE Coordinating Center: Westat, Inc., Rockville, Maryland: O’Brien B; U24 PR:FUNDING_SOURCE ES026539 #STUDY ST:STUDY_TITLE Impacts of interactions between environmental chemical exposures and diet on gut ST:STUDY_TITLE microbiota and microbiota-derived metabolites in mothers and children (MARCH ST:STUDY_TITLE Cohort) ST:STUDY_TYPE Prospective Cohort Study ST:STUDY_SUMMARY MARCH is a prospective population based pregnancy cohort that recruited pregnant ST:STUDY_SUMMARY participants from 2017-2023 from 11 sites within Michigan. Women over 18 years ST:STUDY_SUMMARY of age were recruited at first prenatal visit and had 3 data collection points ST:STUDY_SUMMARY focused around each trimester of pregnancy. Blood, urine, and questionnaire data ST:STUDY_SUMMARY were collected at each prenatal visit. At birth placenta was collected and after ST:STUDY_SUMMARY birth, infants were assessed at 3 months, 9 months, and yearly. For more ST:STUDY_SUMMARY information on MARCH please contact Jean Kerver; kerverje@msu.edu This research ST:STUDY_SUMMARY was supported by the Environmental influences on Child Health Outcomes (ECHO) ST:STUDY_SUMMARY OIF program, Office of The Director, National Institutes of Health. The content ST:STUDY_SUMMARY is solely the responsibility of the authors and does not necessarily represent ST:STUDY_SUMMARY the official views of the National Institutes of Health. MARCH is an ECHO cohort ST:STUDY_SUMMARY which is supported by the following ECHO Program Collaborators: ECHO ST:STUDY_SUMMARY Coordinating Center: Duke Clinical Research Institute, Durham, North Carolina: ST:STUDY_SUMMARY Smith PB, Newby KL, Benjamin DK; U2C OD023375 ECHO Data Analysis Center: Johns ST:STUDY_SUMMARY Hopkins University Bloomberg School of Public Health, Baltimore, Maryland: ST:STUDY_SUMMARY Jacobson LP; Research Triangle Institute, Durham, North Carolina: Catellier.D ST:STUDY_SUMMARY U24 OD023382 North Carolina Human Health Exposure Analysis Resource Hub: ST:STUDY_SUMMARY Research Triangle Institute: Fennell T, University of North Carolina at Chapel ST:STUDY_SUMMARY Hill: Sumner S, University of North Carolina at Charlotte: Du X; U2C ES030857 ST:STUDY_SUMMARY Human Health Exposure Analysis Resource Coordinating Center: Westat, Inc., ST:STUDY_SUMMARY Rockville, Maryland: O’Brien B; U24 ES026539 ST:INSTITUTE Wayne State University ST:DEPARTMENT Pharmacology ST:LAST_NAME Petriello ST:FIRST_NAME Michael ST:ADDRESS 6135 Woodward Ave, Detroit, MI 48202 ST:EMAIL michael.petriello@wayne.edu ST:PHONE 313-577-1089 ST:TOTAL_SUBJECTS 65 ST:STUDY_COMMENTS ECHO Project EC0549, HHEAR Project E020-0012 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 N/A MARCH_HHEAR_1 Sample Type:HHEAR Pool RAW_FILE_NAME=MARCH_HHEAR_1 SUBJECT_SAMPLE_FACTORS N/A MARCH_HHEAR_2 Sample Type:HHEAR Pool RAW_FILE_NAME=MARCH_HHEAR_2 SUBJECT_SAMPLE_FACTORS N/A MARCH_HHEAR_3 Sample Type:HHEAR Pool RAW_FILE_NAME=MARCH_HHEAR_3 SUBJECT_SAMPLE_FACTORS N/A MARCH_HHEAR_4 Sample Type:HHEAR Pool RAW_FILE_NAME=MARCH_HHEAR_4 SUBJECT_SAMPLE_FACTORS N/A MARCH_HHEAR_5 Sample Type:HHEAR Pool RAW_FILE_NAME=MARCH_HHEAR_5 SUBJECT_SAMPLE_FACTORS N/A MARCH_HHEAR_6 Sample Type:HHEAR Pool RAW_FILE_NAME=MARCH_HHEAR_6 SUBJECT_SAMPLE_FACTORS N/A MARCH_HHEAR_7 Sample Type:HHEAR Pool RAW_FILE_NAME=MARCH_HHEAR_7 SUBJECT_SAMPLE_FACTORS N/A MARCH_NIST_1 Sample Type:NIST Pool RAW_FILE_NAME=MARCH_NIST_1 SUBJECT_SAMPLE_FACTORS N/A MARCH_NIST_2 Sample Type:NIST Pool RAW_FILE_NAME=MARCH_NIST_2 SUBJECT_SAMPLE_FACTORS N/A MARCH_NIST_3 Sample Type:NIST Pool RAW_FILE_NAME=MARCH_NIST_3 SUBJECT_SAMPLE_FACTORS EO-37YQ9-P-00-12902 MARCH_S_1 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_1 SUBJECT_SAMPLE_FACTORS EO-38729-P-00-12902 MARCH_S_2 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_2 SUBJECT_SAMPLE_FACTORS EO-38612-P-00-12902 MARCH_S_3 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_3 SUBJECT_SAMPLE_FACTORS EO-37YA4-P-00-12902 MARCH_S_4 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_4 SUBJECT_SAMPLE_FACTORS EO-38BP2-P-00-12902 MARCH_S_5 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_5 SUBJECT_SAMPLE_FACTORS EO-385Y5-P-00-12902 MARCH_S_6 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_6 SUBJECT_SAMPLE_FACTORS EO-37XJ5-P-00-12902 MARCH_S_7 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_7 SUBJECT_SAMPLE_FACTORS EO-389Q9-P-00-12902 MARCH_S_8 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_8 SUBJECT_SAMPLE_FACTORS EO-383F9-P-00-12902 MARCH_S_9 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_9 SUBJECT_SAMPLE_FACTORS EO-37YM8-P-00-12902 MARCH_S_10 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_10 SUBJECT_SAMPLE_FACTORS EO-38EH7-P-00-12902 MARCH_S_11 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_11 SUBJECT_SAMPLE_FACTORS EO-38430-P-00-12902 MARCH_S_12 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_12 SUBJECT_SAMPLE_FACTORS EO-38BQ0-P-00-12902 MARCH_S_13 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_13 SUBJECT_SAMPLE_FACTORS EO-38406-P-00-12902 MARCH_S_14 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_14 SUBJECT_SAMPLE_FACTORS EO-38984-P-00-12902 MARCH_S_15 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_15 SUBJECT_SAMPLE_FACTORS EO-382F0-P-00-12902 MARCH_S_16 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_16 SUBJECT_SAMPLE_FACTORS EO-38885-P-00-12902 MARCH_S_17 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_17 SUBJECT_SAMPLE_FACTORS EO-38182-P-00-12902 MARCH_S_18 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_18 SUBJECT_SAMPLE_FACTORS EO-380N4-P-00-12902 MARCH_S_19 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_19 SUBJECT_SAMPLE_FACTORS EO-387X5-P-00-12902 MARCH_S_20 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_20 SUBJECT_SAMPLE_FACTORS EO-387L2-P-00-12902 MARCH_S_21 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_21 SUBJECT_SAMPLE_FACTORS EO-380P0-P-00-12902 MARCH_S_22 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_22 SUBJECT_SAMPLE_FACTORS EO-384N0-P-00-12902 MARCH_S_23 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_23 SUBJECT_SAMPLE_FACTORS EO-37ZE4-P-00-12902 MARCH_S_24 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_24 SUBJECT_SAMPLE_FACTORS EO-38018-P-00-12902 MARCH_S_25 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_25 SUBJECT_SAMPLE_FACTORS EO-385L4-P-00-12902 MARCH_S_26 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_26 SUBJECT_SAMPLE_FACTORS EO-38CX3-P-00-12902 MARCH_S_27 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_27 SUBJECT_SAMPLE_FACTORS EO-383B8-P-00-12902 MARCH_S_28 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_28 SUBJECT_SAMPLE_FACTORS EO-38EJ2-P-00-12902 MARCH_S_29 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_29 SUBJECT_SAMPLE_FACTORS EO-38BM9-P-00-12902 MARCH_S_30 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_30 SUBJECT_SAMPLE_FACTORS EO-38356-P-00-12902 MARCH_S_31 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_31 SUBJECT_SAMPLE_FACTORS EO-37Z42-P-00-12902 MARCH_S_32 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_32 SUBJECT_SAMPLE_FACTORS EO-38CL0-P-00-12902 MARCH_S_33 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_33 SUBJECT_SAMPLE_FACTORS EO-381Y9-P-00-12902 MARCH_S_34 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_34 SUBJECT_SAMPLE_FACTORS EO-387U2-P-00-12902 MARCH_S_35 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_35 SUBJECT_SAMPLE_FACTORS EO-38BD8-P-00-12902 MARCH_S_36 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_36 SUBJECT_SAMPLE_FACTORS EO-381R5-P-00-12902 MARCH_S_37 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_37 SUBJECT_SAMPLE_FACTORS EO-38BS5-P-00-12902 MARCH_S_38 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_38 SUBJECT_SAMPLE_FACTORS EO-38083-P-00-12902 MARCH_S_39 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_39 SUBJECT_SAMPLE_FACTORS EO-383Q5-P-00-12902 MARCH_S_40 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_40 SUBJECT_SAMPLE_FACTORS EO-38B36-P-00-12902 MARCH_S_41 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_41 SUBJECT_SAMPLE_FACTORS EO-37Y27-P-00-12902 MARCH_S_42 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_42 SUBJECT_SAMPLE_FACTORS EO-383R3-P-00-12902 MARCH_S_43 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_43 SUBJECT_SAMPLE_FACTORS EO-389R7-P-00-12902 MARCH_S_44 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_44 SUBJECT_SAMPLE_FACTORS EO-37YX3-P-00-12902 MARCH_S_45 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_45 SUBJECT_SAMPLE_FACTORS EO-387J6-P-00-12902 MARCH_S_46 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_46 SUBJECT_SAMPLE_FACTORS EO-388U1-P-00-12902 MARCH_S_47 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_47 SUBJECT_SAMPLE_FACTORS EO-38BF4-P-00-12902 MARCH_S_48 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_48 SUBJECT_SAMPLE_FACTORS EO-386Q2-P-00-12902 MARCH_S_49 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_49 SUBJECT_SAMPLE_FACTORS EO-386D0-P-00-12902 MARCH_S_50 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_50 SUBJECT_SAMPLE_FACTORS EO-38794-P-00-12902 MARCH_S_51 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_51 SUBJECT_SAMPLE_FACTORS EO-38075-P-00-12902 MARCH_S_52 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_52 SUBJECT_SAMPLE_FACTORS EO-38BX4-P-00-12902 MARCH_S_53 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_53 SUBJECT_SAMPLE_FACTORS EO-380M7-P-00-12902 MARCH_S_54 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_54 SUBJECT_SAMPLE_FACTORS EO-38A78-P-00-12902 MARCH_S_55 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_55 SUBJECT_SAMPLE_FACTORS EO-37XU1-P-00-12902 MARCH_S_56 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_56 SUBJECT_SAMPLE_FACTORS EO-382T9-P-00-12902 MARCH_S_57 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_57 SUBJECT_SAMPLE_FACTORS EO-38AN7-P-00-12902 MARCH_S_58 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_58 SUBJECT_SAMPLE_FACTORS EO-38DM7-P-00-12902 MARCH_S_59 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_59 SUBJECT_SAMPLE_FACTORS EO-382B9-P-00-12902 MARCH_S_60 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_60 SUBJECT_SAMPLE_FACTORS EO-384A9-P-00-12902 MARCH_S_61 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_61 SUBJECT_SAMPLE_FACTORS EO-385C4-P-00-12902 MARCH_S_62 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_62 SUBJECT_SAMPLE_FACTORS EO-38687-P-00-12902 MARCH_S_63 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_63 SUBJECT_SAMPLE_FACTORS EO-38BC1-P-00-12902 MARCH_S_64 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_64 SUBJECT_SAMPLE_FACTORS EO-38DQ8-P-00-12902 MARCH_S_65 Sample Type:Study Sample RAW_FILE_NAME=MARCH_S_65 SUBJECT_SAMPLE_FACTORS N/A MARCH_SP_1 Sample Type:Study Pool RAW_FILE_NAME=MARCH_SP_1 SUBJECT_SAMPLE_FACTORS N/A MARCH_SP_2 Sample Type:Study Pool RAW_FILE_NAME=MARCH_SP_2 SUBJECT_SAMPLE_FACTORS N/A MARCH_SP_3 Sample Type:Study Pool RAW_FILE_NAME=MARCH_SP_3 SUBJECT_SAMPLE_FACTORS N/A MARCH_SP_4 Sample Type:Study Pool RAW_FILE_NAME=MARCH_SP_4 SUBJECT_SAMPLE_FACTORS N/A MARCH_SP_5 Sample Type:Study Pool RAW_FILE_NAME=MARCH_SP_5 SUBJECT_SAMPLE_FACTORS N/A MARCH_SP_6 Sample Type:Study Pool RAW_FILE_NAME=MARCH_SP_6 SUBJECT_SAMPLE_FACTORS N/A MARCH_SP_7 Sample Type:Study Pool RAW_FILE_NAME=MARCH_SP_7 #COLLECTION CO:COLLECTION_SUMMARY MARCH is a prospective population based pregnancy cohort that recruited pregnant CO:COLLECTION_SUMMARY participants from 2017-2023 from 11 sites within Michigan. Women over 18 years CO:COLLECTION_SUMMARY of age were recruited at first prenatal visit and had 3 data collection points CO:COLLECTION_SUMMARY focused around each trimester of pregnancy. Blood, urine, and questionnaire data CO:COLLECTION_SUMMARY were collected at each prenatal visit. At birth placenta was collected and after CO:COLLECTION_SUMMARY birth, infants were assessed at 3 months, 9 months, and yearly. For more CO:COLLECTION_SUMMARY information on MARCH please contact Jean Kerver; kerverje@msu.edu CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Study samples (500 µL aliquots) were shipped from Wayne State University to the SP:SAMPLEPREP_SUMMARY NC HHEAR Hub on dry ice. The NC HHEAR Hub thawed and aliquoted the study samples SP:SAMPLEPREP_SUMMARY at 50 µL per aliquot. An additional 10 µL was taken from each study sample, SP:SAMPLEPREP_SUMMARY mixed, and distributed at 50 µL per aliquot, which were used as QC study pools SP:SAMPLEPREP_SUMMARY throughout the study. HHEAR reference plasma (50 µL each) and NIST plasma SP:SAMPLEPREP_SUMMARY (1950) reference material (50 µL each) were provided by the NC HHEAR Hub. LC-MS SP:SAMPLEPREP_SUMMARY grade water (50 µL) was used as blank. All samples were thawed at 4°C SP:SAMPLEPREP_SUMMARY overnight before the preparation. Samples, including study samples, study pool SP:SAMPLEPREP_SUMMARY samples, HHEAR reference plasma, NIST reference plasma, and blanks were mixed SP:SAMPLEPREP_SUMMARY with 400 µL methanol containing 500 ng/mL tryptophan-d5 as internal standard SP:SAMPLEPREP_SUMMARY and vortexed by a multiple tube vortex mixer for 2 min at 5000 rpm at room SP:SAMPLEPREP_SUMMARY temperature. All samples were centrifuged at 16,000 rcf for 10 min at 4°C. The SP:SAMPLEPREP_SUMMARY supernatant (350 µL) was transferred into a pre-labeled 2.0 mL Lo-bind SP:SAMPLEPREP_SUMMARY Eppendorf tube, dried by a SpeedVac overnight, and stored at -80° C. For SP:SAMPLEPREP_SUMMARY immediate analysis, 100 µL of water-methanol solution (95:5, v/v) was used to SP:SAMPLEPREP_SUMMARY reconstitute the dried extracts. Samples were thoroughly mixed on a multiple SP:SAMPLEPREP_SUMMARY tube vortex mixer for 10 min at 5000 rpm at room temperature and then SP:SAMPLEPREP_SUMMARY centrifuged at 4°C for 10 min at 16,000 rcf. The supernatant was transferred to SP:SAMPLEPREP_SUMMARY pre-labeled autosampler vials for data acquisition by LC-MS. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Vortex with methanol containing 500ng/ml tryptophan-d5 as internal standard SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Water-Methanol (95:5, v/v) SP:SAMPLE_SPIKING Tryptophan-d5 stock solution at 500 ng/mL #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% methanol; 0.1% formic acid CH:FLOW_GRADIENT Time(min) Flow Rate %A %B Curve 1. 0 0.4 99.0 1.0 5 2. 1.00 0.4 99.0 1.0 5 3. CH:FLOW_GRADIENT 16.00 0.4 1.0 99.0 5 4. 19.00 0.4 1.0 99.0 5 5. 19.50 0.4 99.0 1.0 5 6. 22.00 CH:FLOW_GRADIENT 0.4 99.0 1.0 5 CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 50 CH:COLUMN_PRESSURE 6000-10000 psi CH:INJECTION_TEMPERATURE 8 CH:INTERNAL_STANDARD Tryptophan-d5 CH:ANALYTICAL_TIME 22 min CH:WEAK_WASH_SOLVENT_NAME 10:90 Methanol:Water with 0.1% FA solution CH:STRONG_WASH_SOLVENT_NAME 75:25 2-Propanol:Water with 0.1% FA solution #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Xcalibur 4.1.31.9 for data acquisition; Progenesis QI 2.4 for data preprocessing MS:CAPILLARY_TEMPERATURE 320 °C MS:CAPILLARY_VOLTAGE 3.5 kV MS:COLLISION_ENERGY 20-45, ramp MS:COLLISION_GAS N2 MS:DRY_GAS_TEMP 400°C MS:FRAGMENTATION_METHOD CID MS:DESOLVATION_GAS_FLOW 55 MS:MS_RESULTS_FILE ST002441_AN003978_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END