#METABOLOMICS WORKBENCH wpathmasiri_20230426_090647 DATATRACK_ID:3895 STUDY_ID:ST002692 ANALYSIS_ID:AN004365 VERSION 1 CREATED_ON 09-06-2023 #PROJECT PR:PROJECT_TITLE Per- and poly-fluoroalkyl substances (PFAS) Exposures and Child Health (PEACH) PR:PROJECT_TITLE Study: Using targeted exposure assessment and untargeted metabolic profiling to PR:PROJECT_TITLE characterize molecular pathways and mechanisms underlying PFAS toxicity on PR:PROJECT_TITLE adverse birth and child health outcomes PR:PROJECT_TYPE C18 Reversed-Phase Broad Spectrum Metabolomics PR:PROJECT_SUMMARY The overarching goal of the Per- and poly-fluoroalkyl substances Exposures And PR:PROJECT_SUMMARY Child Health (PEACH) Study is to apply an advanced untargeted metabolomics PR:PROJECT_SUMMARY workflow to investigate associations between PFAS levels, perturbations in PR:PROJECT_SUMMARY maternal and newborn metabolome and adverse birth outcomes. The Emory ECHO team PR:PROJECT_SUMMARY has established a socio-economically diverse, exceptionally phenotyped African PR:PROJECT_SUMMARY American (AA) maternal-child cohort that enrolls pregnant women in the early PR:PROJECT_SUMMARY prenatal period and extends dyad follow-up through age five. PEACH draws from PR:PROJECT_SUMMARY repeated metabolic profiling on a subset of 320 AA pregnant people within the PR:PROJECT_SUMMARY Atlanta ECHO cohort, PFAS assessment, and untargeted metabolomics from newborn PR:PROJECT_SUMMARY blood spots (n=279). Please contact Drs. Donghai Liang (Donghai.liang@emory.edu) PR:PROJECT_SUMMARY and Anne Dunlop (amlang@emory.edu) via email for questions related to the PR:PROJECT_SUMMARY subject characteristics and outcomes. PR:INSTITUTE NC HHEAR Hub PR:DEPARTMENT Untargeted Analysis PR:LABORATORY Sumner Lab PR:LAST_NAME Rushing PR:FIRST_NAME Blake PR:ADDRESS Nutrition Research Institute, UNC-CH, 500 Laureate Way, Kannapolis, NC 28081 PR:EMAIL blake_rushing@unc.edu PR:PHONE (704) 282-9838 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 PEACH 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 PR:DOI http://dx.doi.org/10.21228/M87727 #STUDY ST:STUDY_TITLE Per- and poly-fluoroalkyl substances (PFAS) Exposures and Child Health (PEACH) ST:STUDY_TITLE Study: Using targeted exposure assessment and untargeted metabolic profiling to ST:STUDY_TITLE characterize molecular pathways and mechanisms underlying PFAS toxicity on ST:STUDY_TITLE adverse birth and child health outcomes ST:STUDY_TYPE Prospective Cohort Study ST:STUDY_SUMMARY The overarching goal of the Per- and poly-fluoroalkyl substances Exposures And ST:STUDY_SUMMARY Child Health (PEACH) Study is to apply an advanced untargeted metabolomics ST:STUDY_SUMMARY workflow to investigate associations between PFAS levels, perturbations in ST:STUDY_SUMMARY maternal and newborn metabolome and adverse birth outcomes. The Emory ECHO team ST:STUDY_SUMMARY has established a socio-economically diverse, exceptionally phenotyped African ST:STUDY_SUMMARY American (AA) maternal-child cohort that enrolls pregnant women in the early ST:STUDY_SUMMARY prenatal period and extends dyad follow-up through age five. PEACH draws from ST:STUDY_SUMMARY repeated metabolic profiling on a subset of 320 AA pregnant people within the ST:STUDY_SUMMARY Atlanta ECHO cohort, PFAS assessment, and untargeted metabolomics from newborn ST:STUDY_SUMMARY blood spots (n=279). Please contact Drs. Donghai Liang (Donghai.liang@emory.edu) ST:STUDY_SUMMARY and Anne Dunlop (amlang@emory.edu) via email for questions related to the ST:STUDY_SUMMARY subject characteristics and outcomes. This research was supported by the ST:STUDY_SUMMARY Environmental influences on Child Health Outcomes (ECHO) OIF program, Office of ST:STUDY_SUMMARY The Director, National Institutes of Health. The content is solely the ST:STUDY_SUMMARY responsibility of the authors and does not necessarily represent the official ST:STUDY_SUMMARY views of the National Institutes of Health. PEACH is an ECHO cohort which is ST:STUDY_SUMMARY supported by the following ECHO Program Collaborators: ECHO Coordinating Center: ST:STUDY_SUMMARY Duke Clinical Research Institute, Durham, North Carolina: Smith PB, Newby KL, ST:STUDY_SUMMARY Benjamin DK; U2C OD023375 ECHO Data Analysis Center: Johns Hopkins University ST:STUDY_SUMMARY Bloomberg School of Public Health, Baltimore, Maryland: Jacobson LP; Research ST:STUDY_SUMMARY Triangle Institute, Durham, North Carolina: Catellier D; U24 OD023382 North ST:STUDY_SUMMARY Carolina Human Health Exposure Analysis Resource Hub: Research Triangle ST:STUDY_SUMMARY Institute: Fennell T, University of North Carolina at Chapel Hill: Sumner S, ST:STUDY_SUMMARY University of North Carolina at Charlotte: Du X; U2C ES030857 Human Health ST:STUDY_SUMMARY Exposure Analysis Resource Coordinating Center: Westat, Inc., Rockville, ST:STUDY_SUMMARY Maryland: O’Brien B; U24 ES026539 ST:INSTITUTE Emory University ST:DEPARTMENT Environmental Health ST:LAST_NAME Liang ST:FIRST_NAME Donghai ST:ADDRESS 1518 Clifton Rd. NE Atlanta, GA 30322 ST:EMAIL donghai.liang@emory.edu ST:PHONE (404) 712-9583 ST:SUBMIT_DATE 2023-04-26 #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]Additional sample data SUBJECT_SAMPLE_FACTORS - BP_1 Sample Type:Blank | Batch:1 Analysis Order=11; RAW_FILE_NAME=BP_1 SUBJECT_SAMPLE_FACTORS - BP_2 Sample Type:Blank | Batch:1 Analysis Order=24; RAW_FILE_NAME=BP_2 SUBJECT_SAMPLE_FACTORS - BP_3 Sample Type:Blank | Batch:1 Analysis Order=36; RAW_FILE_NAME=BP_3 SUBJECT_SAMPLE_FACTORS - BP_4 Sample Type:Blank | Batch:1 Analysis Order=49; RAW_FILE_NAME=BP_4 SUBJECT_SAMPLE_FACTORS - BP_5 Sample Type:Blank | Batch:1 Analysis Order=62; RAW_FILE_NAME=BP_5 SUBJECT_SAMPLE_FACTORS - BP_6 Sample Type:Blank | Batch:1 Analysis Order=75; RAW_FILE_NAME=BP_6 SUBJECT_SAMPLE_FACTORS - BP_7 Sample Type:Blank | Batch:1 Analysis Order=88; RAW_FILE_NAME=BP_7 SUBJECT_SAMPLE_FACTORS - BP_8 Sample Type:Blank | Batch:1 Analysis Order=101; 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RAW_FILE_NAME=S_210 SUBJECT_SAMPLE_FACTORS G0187-NBS_T1 S_211 Sample Type:Study sample | Batch:3 Analysis Order=273; RAW_FILE_NAME=S_211 SUBJECT_SAMPLE_FACTORS E0645-NBS_T1 S_212 Sample Type:Study sample | Batch:3 Analysis Order=274; RAW_FILE_NAME=S_212 SUBJECT_SAMPLE_FACTORS E0668-NBS_T1 S_213 Sample Type:Study sample | Batch:3 Analysis Order=275; RAW_FILE_NAME=S_213 SUBJECT_SAMPLE_FACTORS G0285-NBS_T1 S_214 Sample Type:Study sample | Batch:3 Analysis Order=276; RAW_FILE_NAME=S_214 SUBJECT_SAMPLE_FACTORS G0301-NBS_T1 S_215 Sample Type:Study sample | Batch:3 Analysis Order=277; RAW_FILE_NAME=S_215 SUBJECT_SAMPLE_FACTORS G0389-NBS_T1 S_216 Sample Type:Study sample | Batch:3 Analysis Order=278; RAW_FILE_NAME=S_216 SUBJECT_SAMPLE_FACTORS G0266-NBS_T1 S_217 Sample Type:Study sample | Batch:3 Analysis Order=279; RAW_FILE_NAME=S_217 SUBJECT_SAMPLE_FACTORS G0309-NBS_T1 S_218 Sample Type:Study sample | Batch:3 Analysis Order=280; RAW_FILE_NAME=S_218 SUBJECT_SAMPLE_FACTORS E0589-NBS_T1 S_219 Sample Type:Study sample | Batch:3 Analysis Order=281; RAW_FILE_NAME=S_219 SUBJECT_SAMPLE_FACTORS G0245-NBS_T1 S_220 Sample Type:Study sample | Batch:3 Analysis Order=282; RAW_FILE_NAME=S_220 SUBJECT_SAMPLE_FACTORS E0641-NBS_T1 S_221 Sample Type:Study sample | Batch:3 Analysis Order=286; RAW_FILE_NAME=S_221 SUBJECT_SAMPLE_FACTORS G0146-NBS_T1 S_222 Sample Type:Study sample | Batch:3 Analysis Order=287; RAW_FILE_NAME=S_222 SUBJECT_SAMPLE_FACTORS G0291-NBS_T1 S_223 Sample Type:Study sample | Batch:3 Analysis Order=288; RAW_FILE_NAME=S_223 SUBJECT_SAMPLE_FACTORS G0204-NBS_T1 S_224 Sample Type:Study sample | Batch:3 Analysis Order=289; RAW_FILE_NAME=S_224 SUBJECT_SAMPLE_FACTORS G0253-NBS_T1 S_225 Sample Type:Study sample | Batch:3 Analysis Order=290; RAW_FILE_NAME=S_225 SUBJECT_SAMPLE_FACTORS G0161-NBS_T1 S_226 Sample Type:Study sample | Batch:3 Analysis Order=291; RAW_FILE_NAME=S_226 SUBJECT_SAMPLE_FACTORS G0354-NBS_T1 S_227 Sample Type:Study sample | Batch:3 Analysis Order=292; RAW_FILE_NAME=S_227 SUBJECT_SAMPLE_FACTORS G0164-NBS_T1 S_228 Sample Type:Study sample | Batch:3 Analysis Order=293; RAW_FILE_NAME=S_228 SUBJECT_SAMPLE_FACTORS E0691-NBS_T1 S_229 Sample Type:Study sample | Batch:3 Analysis Order=294; RAW_FILE_NAME=S_229 SUBJECT_SAMPLE_FACTORS G0333-NBS_T1 S_230 Sample Type:Study sample | Batch:3 Analysis Order=295; RAW_FILE_NAME=S_230 SUBJECT_SAMPLE_FACTORS E0700-NBS_T1 S_231 Sample Type:Study sample | Batch:3 Analysis Order=299; RAW_FILE_NAME=S_231 SUBJECT_SAMPLE_FACTORS E0669-NBS_T1 S_232 Sample Type:Study sample | Batch:3 Analysis Order=300; RAW_FILE_NAME=S_232 SUBJECT_SAMPLE_FACTORS E0636-NBS_T1 S_233 Sample Type:Study sample | Batch:3 Analysis Order=301; RAW_FILE_NAME=S_233 SUBJECT_SAMPLE_FACTORS E0637-NBS_T1 S_234 Sample Type:Study sample | Batch:3 Analysis Order=302; RAW_FILE_NAME=S_234 SUBJECT_SAMPLE_FACTORS G0255-NBS_T1 S_235 Sample Type:Study sample | Batch:3 Analysis Order=303; RAW_FILE_NAME=S_235 SUBJECT_SAMPLE_FACTORS G0244-NBS_T1 S_236 Sample Type:Study sample | Batch:3 Analysis Order=304; RAW_FILE_NAME=S_236 SUBJECT_SAMPLE_FACTORS E0621-NBS_T1 S_237 Sample Type:Study sample | Batch:3 Analysis Order=305; RAW_FILE_NAME=S_237 SUBJECT_SAMPLE_FACTORS E0665-NBS_T1 S_238 Sample Type:Study sample | Batch:3 Analysis Order=306; RAW_FILE_NAME=S_238 SUBJECT_SAMPLE_FACTORS G0151-NBS_T1 S_239 Sample Type:Study sample | Batch:3 Analysis Order=307; RAW_FILE_NAME=S_239 SUBJECT_SAMPLE_FACTORS E0627-NBS_T1 S_240 Sample Type:Study sample | Batch:3 Analysis Order=308; RAW_FILE_NAME=S_240 SUBJECT_SAMPLE_FACTORS G0365-NBS_T1 S_241 Sample Type:Study sample | Batch:3 Analysis Order=312; RAW_FILE_NAME=S_241 SUBJECT_SAMPLE_FACTORS E0611-NBS_T1 S_242 Sample Type:Study sample | Batch:3 Analysis Order=313; RAW_FILE_NAME=S_242 SUBJECT_SAMPLE_FACTORS E0758-NBS_T1 S_243 Sample Type:Study sample | Batch:3 Analysis Order=314; RAW_FILE_NAME=S_243 SUBJECT_SAMPLE_FACTORS E0610-NBS_T1 S_244 Sample Type:Study sample | Batch:3 Analysis Order=315; RAW_FILE_NAME=S_244 SUBJECT_SAMPLE_FACTORS G0347-NBS_T1 S_245 Sample Type:Study sample | Batch:3 Analysis Order=316; RAW_FILE_NAME=S_245 SUBJECT_SAMPLE_FACTORS G0232-NBS_T1 S_246 Sample Type:Study sample | Batch:3 Analysis Order=317; RAW_FILE_NAME=S_246 SUBJECT_SAMPLE_FACTORS G0265-NBS_T1 S_247 Sample Type:Study sample | Batch:3 Analysis Order=318; RAW_FILE_NAME=S_247 SUBJECT_SAMPLE_FACTORS G0335-NBS_T1 S_248 Sample Type:Study sample | Batch:3 Analysis Order=319; RAW_FILE_NAME=S_248 SUBJECT_SAMPLE_FACTORS G0313-NBS_T1 S_249 Sample Type:Study sample | Batch:3 Analysis Order=320; RAW_FILE_NAME=S_249 SUBJECT_SAMPLE_FACTORS G0353-NBS_T1 S_250 Sample Type:Study sample | Batch:3 Analysis Order=321; RAW_FILE_NAME=S_250 SUBJECT_SAMPLE_FACTORS G0346-NBS_T1 S_251 Sample Type:Study sample | Batch:3 Analysis Order=325; RAW_FILE_NAME=S_251 SUBJECT_SAMPLE_FACTORS G0254-NBS_T1 S_252 Sample Type:Study sample | Batch:3 Analysis Order=326; RAW_FILE_NAME=S_252 SUBJECT_SAMPLE_FACTORS E0624-NBS_T1 S_253 Sample Type:Study sample | Batch:3 Analysis Order=327; RAW_FILE_NAME=S_253 SUBJECT_SAMPLE_FACTORS E0692-NBS_T1 S_254 Sample Type:Study sample | Batch:3 Analysis Order=328; RAW_FILE_NAME=S_254 SUBJECT_SAMPLE_FACTORS G0221-NBS_T1 S_255 Sample Type:Study sample | Batch:3 Analysis Order=329; RAW_FILE_NAME=S_255 SUBJECT_SAMPLE_FACTORS G0267-NBS_T1 S_256 Sample Type:Study sample | Batch:3 Analysis Order=330; RAW_FILE_NAME=S_256 SUBJECT_SAMPLE_FACTORS G0209-NBS_T1 S_257 Sample Type:Study sample | Batch:3 Analysis Order=331; RAW_FILE_NAME=S_257 SUBJECT_SAMPLE_FACTORS G0163-NBS_T1 S_258 Sample Type:Study sample | Batch:3 Analysis Order=332; RAW_FILE_NAME=S_258 SUBJECT_SAMPLE_FACTORS G0193-NBS_T1 S_259 Sample Type:Study sample | Batch:3 Analysis Order=333; RAW_FILE_NAME=S_259 SUBJECT_SAMPLE_FACTORS G0379-NBS_T1 S_260 Sample Type:Study sample | Batch:3 Analysis Order=334; RAW_FILE_NAME=S_260 SUBJECT_SAMPLE_FACTORS G0183-NBS_T1 S_261 Sample Type:Study sample | Batch:3 Analysis Order=338; RAW_FILE_NAME=S_261 SUBJECT_SAMPLE_FACTORS E0710-NBS_T1 S_262 Sample Type:Study sample | Batch:3 Analysis Order=339; RAW_FILE_NAME=S_262 SUBJECT_SAMPLE_FACTORS G0368-NBS_T1 S_263 Sample Type:Study sample | Batch:3 Analysis Order=340; RAW_FILE_NAME=S_263 SUBJECT_SAMPLE_FACTORS G0125-NBS_T1 S_264 Sample Type:Study sample | Batch:3 Analysis Order=341; RAW_FILE_NAME=S_264 SUBJECT_SAMPLE_FACTORS G0358-NBS_T1 S_265 Sample Type:Study sample | Batch:3 Analysis Order=342; RAW_FILE_NAME=S_265 SUBJECT_SAMPLE_FACTORS G0273-NBS_T1 S_266 Sample Type:Study sample | Batch:3 Analysis Order=343; RAW_FILE_NAME=S_266 SUBJECT_SAMPLE_FACTORS G0184-NBS_T1 S_267 Sample Type:Study sample | Batch:3 Analysis Order=344; RAW_FILE_NAME=S_267 SUBJECT_SAMPLE_FACTORS G0190-NBS_T1 S_268 Sample Type:Study sample | Batch:3 Analysis Order=345; RAW_FILE_NAME=S_268 SUBJECT_SAMPLE_FACTORS G0401-NBS_T1 S_269 Sample Type:Study sample | Batch:3 Analysis Order=346; RAW_FILE_NAME=S_269 SUBJECT_SAMPLE_FACTORS G0124-NBS_T1 S_270 Sample Type:Study sample | Batch:3 Analysis Order=347; RAW_FILE_NAME=S_270 SUBJECT_SAMPLE_FACTORS G0234-NBS_T1 S_271 Sample Type:Study sample | Batch:3 Analysis Order=351; RAW_FILE_NAME=S_271 SUBJECT_SAMPLE_FACTORS G0282-NBS_T1 S_272 Sample Type:Study sample | Batch:3 Analysis Order=352; RAW_FILE_NAME=S_272 SUBJECT_SAMPLE_FACTORS E0619-NBS_T1 S_273 Sample Type:Study sample | Batch:3 Analysis Order=353; RAW_FILE_NAME=S_273 SUBJECT_SAMPLE_FACTORS G0304-NBS_T1 S_274 Sample Type:Study sample | Batch:3 Analysis Order=354; RAW_FILE_NAME=S_274 SUBJECT_SAMPLE_FACTORS E0750-NBS_T1 S_275 Sample Type:Study sample | Batch:3 Analysis Order=355; RAW_FILE_NAME=S_275 SUBJECT_SAMPLE_FACTORS G0292-NBS_T1 S_276 Sample Type:Study sample | Batch:3 Analysis Order=356; RAW_FILE_NAME=S_276 SUBJECT_SAMPLE_FACTORS G0207-NBS_T1 S_277 Sample Type:Study sample | Batch:3 Analysis Order=357; RAW_FILE_NAME=S_277 SUBJECT_SAMPLE_FACTORS E0656-NBS_T1 S_278 Sample Type:Study sample | Batch:3 Analysis Order=358; RAW_FILE_NAME=S_278 SUBJECT_SAMPLE_FACTORS E0634-NBS_T1 S_279 Sample Type:Study sample | Batch:3 Analysis Order=359; RAW_FILE_NAME=S_279 SUBJECT_SAMPLE_FACTORS E0646-NBS_T1 S_280 Sample Type:Study sample | Batch:3 Analysis Order=360; RAW_FILE_NAME=S_280 #COLLECTION CO:COLLECTION_SUMMARY N/A CO:SAMPLE_TYPE Blood (whole) CO:COLLECTION_METHOD Dried Blood Spot CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY NA #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 279 newborn dried blood spot and blank samples and were randomized and extracted SP:SAMPLEPREP_SUMMARY by 1000 µL methanol with 500 ng/mLtryptophan-d5 as internal standard via a SP:SAMPLEPREP_SUMMARY multiple tube vortex mixer for 10 minutes at 5000 rpm. The blank and study SP:SAMPLEPREP_SUMMARY samples were sonicated in an ice bath for 20 minutes. A volume of 700 µL of the SP:SAMPLEPREP_SUMMARY supernatants was transferred into a pre-labeled 2.0 mL Low-bind Eppendorf tube SP:SAMPLEPREP_SUMMARY labeled as the stock. Protein precipitate was pelleted using a centrifuge SP:SAMPLEPREP_SUMMARY operating at 4° C for 5 minutes at 16,000 rcf. A volume of 70 µL of each blank SP:SAMPLEPREP_SUMMARY sample stock was transferred to 15 mL conical tube to be combined to create a SP:SAMPLEPREP_SUMMARY Blank Pool (BP). A volume of 70 µL of each study sample stock was transferred SP:SAMPLEPREP_SUMMARY to 15 mL conical tube to be combined and create a Study Pool (SP). A total SP:SAMPLEPREP_SUMMARY volume of 1400 µL of NIST Reference Plasma (NIST) was aliquoted into SP:SAMPLEPREP_SUMMARY twenty-eight 50 µL aliquots. A volume of 600 µL supernatant (including SP:SAMPLEPREP_SUMMARY experimental samples, BP, and SP) was transferred into a pre-labeled 2.0 mL SP:SAMPLEPREP_SUMMARY Low-bind Eppendorf tube. All samples and pools were then dried by a SpeedVac SP:SAMPLEPREP_SUMMARY overnight. For immediate analysis, 100 µL of water-methanol solution (95:5, SP:SAMPLEPREP_SUMMARY v/v) was added to reconstitute the dried extracts, and the samples were SP:SAMPLEPREP_SUMMARY thoroughly mixed on multiple tube vortex mixer for 10 min at 5000 rpm. Samples SP:SAMPLEPREP_SUMMARY were centrifuged at 4°C for 4 min at 16,000 rcf. The supernatant was SP:SAMPLEPREP_SUMMARY transferred to pre-labeled autosampler vials for data acquisition by LC-MS. SP:SAMPLEPREP_SUMMARY Broad-spectrum metabolomics was conducted using a Thermo Scientific™ SP:SAMPLEPREP_SUMMARY Vanquish™ UPHPLC - Q Exactive™ HF-X Orbitrap System. Metabolites were SP:SAMPLEPREP_SUMMARY separated on an Acquity UPLC HSS T3 C18 (2.1 X 100 mm, 1.8 µm) operating at 50 SP:SAMPLEPREP_SUMMARY C using a reversed phase gradient separation with Water with 0.1% Formic Acid SP:SAMPLEPREP_SUMMARY (v/v) as mobile phase A and Methanol with 0.1% Formic Acid (v/v) as mobile phase SP:SAMPLEPREP_SUMMARY B. A 5 µL was injected into the instrument, and MS was collected between 50-750 SP:SAMPLEPREP_SUMMARY m/z in positive mode with the MS/MS data triggered by the Data-Dependent SP:SAMPLEPREP_SUMMARY Acquisition. SP:PROCESSING_STORAGE_CONDITIONS 4℃ 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_SUMMARY Reverse phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:COLUMN_PRESSURE 6000-10000 psi CH:COLUMN_TEMPERATURE 50℃ 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:INJECTION_TEMPERATURE 8℃ CH:INTERNAL_STANDARD Tryptophan-d5 CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% methanol; 0.1% formic acid CH:ANALYTICAL_TIME 22 min CH:WEAK_WASH_SOLVENT_NAME 10% methanol/90% water; 0.1% formic acid CH:STRONG_WASH_SOLVENT_NAME 75% 2-Propanol/25% Water; 0.1% formic acid CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:LABORATORY_NAME Sumner Lab, UNC-CH AN:ANALYSIS_TYPE MS AN:ACQUISITION_DATE 12/7/2022-12/12/2022 AN:SOFTWARE_VERSION Xcalibur 4.1.31.9 AN:DETECTOR_TYPE Orbitrap AN:DATA_FORMAT Profile #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Instrument: Thermo Q Exactive HFx Software: Xcalibur 4.1.31.9 for data MS:MS_COMMENTS acquisition; Progenesis QI 2.4 for data preprocessing MS:ION_MODE POSITIVE MS:CAPILLARY_TEMPERATURE 320 °C MS:CAPILLARY_VOLTAGE 3.5 KV MS:COLLISION_ENERGY 20-45, ramp MS:COLLISION_GAS N2 MS:DRY_GAS_FLOW 55 MS:DRY_GAS_TEMP 400°C MS:FRAGMENTATION_METHOD CID MS:ION_SPRAY_VOLTAGE 3.5kV MS:IONIZATION ES+ MS:MASS_ACCURACY 5 ppm MS:DATAFORMAT Profile MS:DESOLVATION_GAS_FLOW 55 MS:DESOLVATION_TEMPERATURE 400 ℃ MS:MS_RESULTS_FILE ST002692_AN004365_Results.txt UNITS:Normalized intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END