#METABOLOMICS WORKBENCH douglas_walker_20200622_081712_mwtab.txt DATATRACK_ID:2060 STUDY_ID:ST001407 ANALYSIS_ID:AN002350 PROJECT_ID:000000 VERSION 1 CREATED_ON June 22, 2020, 12:08 pm #PROJECT PR:PROJECT_TITLE Environmental chemical burden in metabolic tissues and systemic biological PR:PROJECT_TITLE pathways in adolescent bariatric surgery patients: A pilot untargeted PR:PROJECT_TITLE metabolomic approach PR:PROJECT_TYPE Pilot Study PR:PROJECT_SUMMARY Background: Advances in untargeted metabolomic technologies have great potential PR:PROJECT_SUMMARY for insight into adverse metabolic effects underlying exposure to environmental PR:PROJECT_SUMMARY chemicals. However, important challenges need to be addressed, including how PR:PROJECT_SUMMARY biological response corresponds to the environmental chemical burden in PR:PROJECT_SUMMARY different target tissues. Aim: We performed a pilot study using state-of-the-art PR:PROJECT_SUMMARY ultra-high-resolution mass spectrometry (UHRMS) to characterize the burden of PR:PROJECT_SUMMARY lipophilic persistent organic pollutants (POPs) in metabolic tissues and PR:PROJECT_SUMMARY associated alterations in the plasma metabolome. Methods: We studied 11 PR:PROJECT_SUMMARY adolescents with severe obesity at the time of bariatric surgery. We measured 18 PR:PROJECT_SUMMARY POPs that can act as endocrine and metabolic disruptors (i.e. 2 dioxins, 11 PR:PROJECT_SUMMARY organochlorine compounds [OCs] and 5 polybrominated diphenyl ethers [PBDEs]) in PR:PROJECT_SUMMARY visceral and subcutaneous abdominal adipose tissue (vAT and sAT), and liver PR:PROJECT_SUMMARY samples using gas chromatography with UHRMS. Biological pathways were evaluated PR:PROJECT_SUMMARY by measuring the plasma metabolome using high-resolution metabolomics. Network PR:PROJECT_SUMMARY and pathway enrichment analysis assessed correlations between the PR:PROJECT_SUMMARY tissue-specific burden of three frequently detected POPs (i.e. PR:PROJECT_SUMMARY p,p’-dichlorodiphenyldichloroethene [DDE], hexachlorobenzene [HCB] and PR:PROJECT_SUMMARY PBDE-47) and plasma metabolic pathways. Results: Concentrations of 4 OCs and 3 PR:PROJECT_SUMMARY PBDEs were quantifiable in at least one metabolic tissue for >80% of PR:PROJECT_SUMMARY participants. All POPs had the highest median concentrations in adipose tissue, PR:PROJECT_SUMMARY especially sAT, except for PBDE-154, which had comparable average concentrations PR:PROJECT_SUMMARY across all tissues. Pathway analysis showed high correlations between PR:PROJECT_SUMMARY tissue-specific POPs and metabolic alterations in pathways of amino acid PR:PROJECT_SUMMARY metabolism, lipid and fatty acid metabolism, and carbohydrate metabolism. PR:PROJECT_SUMMARY Conclusions: Most of the measured POPs appear to accumulate preferentially in PR:PROJECT_SUMMARY adipose tissue compared to liver. Findings of plasma metabolic pathways PR:PROJECT_SUMMARY potentially associated with tissue-specific POPs concentrations merit further PR:PROJECT_SUMMARY investigation in larger populations. Keywords: persistent organic pollutants, PR:PROJECT_SUMMARY adipose tissue, liver, bariatric surgery, exposome, high-resolution metabolomics PR:INSTITUTE Icahn School of Medicine at Mount Sinai PR:DEPARTMENT Environmental Medicine and Public Health PR:LABORATORY High Resolution Exposomics Research Group PR:LAST_NAME Walker PR:FIRST_NAME Douglas PR:ADDRESS One Gustave L. Levy Place, Box 1057, New York, NY 10029 PR:EMAIL douglas.walker@mssm.edu PR:PHONE 212-241-9891 PR:FUNDING_SOURCE NIEHS: R21ES028903, R21ES029328, R21ES029681, R01ES029944, R01ES030364, PR:FUNDING_SOURCE U2CES026561, U2CES030163, P30ES023515, P30 ES019776, P30ES007048, P01ES022845, PR:FUNDING_SOURCE R01ES024946; EPA: RD-83544101 PR:PROJECT_COMMENTS This upload is 2 of 2 and includes untargeted plasma metabolomics results. Part PR:PROJECT_COMMENTS 1 included targeted levels of organic pollutants PR:PUBLICATIONS Valvi D, Walker DI, Inge T, Bartell SM, Jenkins T, Helmrath M, Ziegler TR, La PR:PUBLICATIONS Merrill MA, Eckel SP, Conti D, Liang Y, Jones DP, McConnell R, Chatzi L. (2020). PR:PUBLICATIONS Environmental chemical burden in metabolic tissues and systemic biological PR:PUBLICATIONS pathways in adolescent bariatric surgery patients: A pilot untargeted PR:PUBLICATIONS metabolomic approach. Environment International. In Press. PR:CONTRIBUTORS Valvi D, Walker DI, Inge T, Bartell SM, Jenkins T, Helmrath M, Ziegler TR, La PR:CONTRIBUTORS Merrill MA, Eckel SP, Conti D, Liang Y, Jones DP, McConnell R, Chatzi L #STUDY ST:STUDY_TITLE Environmental chemical burden in metabolic tissues and systemic biological ST:STUDY_TITLE pathways in adolescent bariatric surgery patients: A pilot untargeted ST:STUDY_TITLE metabolomic approach ST:STUDY_TYPE Subcutaneous adipose tissue (AT); Visceral AT; Liver Tissue; Plasma ST:STUDY_SUMMARY Background: Advances in untargeted metabolomic technologies have great potential ST:STUDY_SUMMARY for insight into adverse metabolic effects underlying exposure to environmental ST:STUDY_SUMMARY chemicals. However, important challenges need to be addressed, including how ST:STUDY_SUMMARY biological response corresponds to the environmental chemical burden in ST:STUDY_SUMMARY different target tissues. Aim: We performed a pilot study using state-of-the-art ST:STUDY_SUMMARY ultra-high-resolution mass spectrometry (UHRMS) to characterize the burden of ST:STUDY_SUMMARY lipophilic persistent organic pollutants (POPs) in metabolic tissues and ST:STUDY_SUMMARY associated alterations in the plasma metabolome. Methods: We studied 11 ST:STUDY_SUMMARY adolescents with severe obesity at the time of bariatric surgery. We measured 18 ST:STUDY_SUMMARY POPs that can act as endocrine and metabolic disruptors (i.e. 2 dioxins, 11 ST:STUDY_SUMMARY organochlorine compounds [OCs] and 5 polybrominated diphenyl ethers [PBDEs]) in ST:STUDY_SUMMARY visceral and subcutaneous abdominal adipose tissue (vAT and sAT), and liver ST:STUDY_SUMMARY samples using gas chromatography with UHRMS. Biological pathways were evaluated ST:STUDY_SUMMARY by measuring the plasma metabolome using high-resolution metabolomics. Network ST:STUDY_SUMMARY and pathway enrichment analysis assessed correlations between the ST:STUDY_SUMMARY tissue-specific burden of three frequently detected POPs (i.e. ST:STUDY_SUMMARY p,p’-dichlorodiphenyldichloroethene [DDE], hexachlorobenzene [HCB] and ST:STUDY_SUMMARY PBDE-47) and plasma metabolic pathways. Results: Concentrations of 4 OCs and 3 ST:STUDY_SUMMARY PBDEs were quantifiable in at least one metabolic tissue for >80% of ST:STUDY_SUMMARY participants. All POPs had the highest median concentrations in adipose tissue, ST:STUDY_SUMMARY especially sAT, except for PBDE-154, which had comparable average concentrations ST:STUDY_SUMMARY across all tissues. Pathway analysis showed high correlations between ST:STUDY_SUMMARY tissue-specific POPs and metabolic alterations in pathways of amino acid ST:STUDY_SUMMARY metabolism, lipid and fatty acid metabolism, and carbohydrate metabolism. ST:STUDY_SUMMARY Conclusions: Most of the measured POPs appear to accumulate preferentially in ST:STUDY_SUMMARY adipose tissue compared to liver. Findings of plasma metabolic pathways ST:STUDY_SUMMARY potentially associated with tissue-specific POPs concentrations merit further ST:STUDY_SUMMARY investigation in larger populations. ST:INSTITUTE Icahn School of Medicine at Mount Sinai ST:DEPARTMENT Environmental Medicine and Public Health ST:LABORATORY High Resolution Exposomics Research Group ST:LAST_NAME Walker ST:FIRST_NAME Doug ST:ADDRESS One Gustave L. Levy Place, Box 1057, New York, NY 10029 ST:EMAIL douglas.walker@mssm.edu ST:PHONE 212-241-9891 ST:NUM_GROUPS 1 ST:TOTAL_SUBJECTS 11 ST:NUM_MALES 1 ST:NUM_FEMALES 10 ST:STUDY_COMMENTS Upload #1: Visceral and subcutaneous abdominal adipose tissue, liver tissue. ST:STUDY_COMMENTS Plasma metabolomics are in upload #2 ST:PUBLICATIONS Valvi D, Walker DI, Inge T, Bartell SM, Jenkins T, Helmrath M, Ziegler TR, La ST:PUBLICATIONS Merrill MA, Eckel SP, Conti D, Liang Y, Jones DP, McConnell R, Chatzi L. (2020). ST:PUBLICATIONS Environmental chemical burden in metabolic tissues and systemic biological ST:PUBLICATIONS pathways in adolescent bariatric surgery patients: A pilot untargeted ST:PUBLICATIONS metabolomic approach. Environment International. In Press. #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 11-20 years SU:GENDER Male and female #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 QC NIST_1958_1 Description:NIST 1958 Batch=1; RAW_FILE_NAME=VT_170706_001; RAW_FILE_NAME=VT_170706_003; RAW_FILE_NAME=VT_170706_005; RAW_FILE_NAME=VT_170706_002; RAW_FILE_NAME=VT_170706_004; RAW_FILE_NAME=VT_170706_006 SUBJECT_SAMPLE_FACTORS QC q3June2014_1a Description:Q-Standard plasma pool Batch=1; RAW_FILE_NAME=VT_170706_007; RAW_FILE_NAME=VT_170706_009; RAW_FILE_NAME=VT_170706_011; RAW_FILE_NAME=VT_170706_008; RAW_FILE_NAME=VT_170706_010; RAW_FILE_NAME=VT_170706_012 SUBJECT_SAMPLE_FACTORS QC q3June2014_1b Description:Q-Standard plasma pool Batch=1; RAW_FILE_NAME=VT_170706_013; RAW_FILE_NAME=VT_170706_015; RAW_FILE_NAME=VT_170706_017; RAW_FILE_NAME=VT_170706_014; RAW_FILE_NAME=VT_170706_016; RAW_FILE_NAME=VT_170706_013 SUBJECT_SAMPLE_FACTORS QC chearplasma_1a Description:CHEAR plasma pool Batch=1; RAW_FILE_NAME=VT_170706_019; RAW_FILE_NAME=VT_170706_021; RAW_FILE_NAME=VT_170706_023; RAW_FILE_NAME=VT_170706_020; RAW_FILE_NAME=VT_170706_022; RAW_FILE_NAME=VT_170706_014 SUBJECT_SAMPLE_FACTORS QC chearplasma_1b Description:CHEAR plasma pool Batch=1; RAW_FILE_NAME=VT_170706_025; RAW_FILE_NAME=VT_170706_027; RAW_FILE_NAME=VT_170706_029; RAW_FILE_NAME=VT_170706_026; RAW_FILE_NAME=VT_170706_028; RAW_FILE_NAME=VT_170706_015 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_02_Plasma Description:POTR_02 Batch=1; RAW_FILE_NAME=VT_170706_037; RAW_FILE_NAME=VT_170706_039; RAW_FILE_NAME=VT_170706_041; RAW_FILE_NAME=VT_170706_038; RAW_FILE_NAME=VT_170706_040; RAW_FILE_NAME=VT_170706_017 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_03_Plasma Description:POTR_03 Batch=1; RAW_FILE_NAME=VT_170706_043; RAW_FILE_NAME=VT_170706_045; RAW_FILE_NAME=VT_170706_047; RAW_FILE_NAME=VT_170706_044; RAW_FILE_NAME=VT_170706_046; RAW_FILE_NAME=VT_170706_018 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_04_Plasma Description:POTR_04 Batch=1; RAW_FILE_NAME=VT_170706_049; RAW_FILE_NAME=VT_170706_051; RAW_FILE_NAME=VT_170706_053; RAW_FILE_NAME=VT_170706_050; RAW_FILE_NAME=VT_170706_052; RAW_FILE_NAME=VT_170706_019 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_05_Plasma Description:POTR_05 Batch=1; RAW_FILE_NAME=VT_170706_055; RAW_FILE_NAME=VT_170706_057; RAW_FILE_NAME=VT_170706_059; RAW_FILE_NAME=VT_170706_056; RAW_FILE_NAME=VT_170706_058; RAW_FILE_NAME=VT_170706_020 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_06_Plasma Description:POTR_06 Batch=1; RAW_FILE_NAME=VT_170706_061; RAW_FILE_NAME=VT_170706_063; RAW_FILE_NAME=VT_170706_065; RAW_FILE_NAME=VT_170706_062; RAW_FILE_NAME=VT_170706_064; RAW_FILE_NAME=VT_170706_021 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_07_Plasma Description:POTR_07 Batch=1; RAW_FILE_NAME=VT_170706_067; RAW_FILE_NAME=VT_170706_069; RAW_FILE_NAME=VT_170706_071; RAW_FILE_NAME=VT_170706_068; RAW_FILE_NAME=VT_170706_070; RAW_FILE_NAME=VT_170706_022 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_08_Plasma Description:POTR_08 Batch=1; RAW_FILE_NAME=VT_170706_073; RAW_FILE_NAME=VT_170706_075; RAW_FILE_NAME=VT_170706_077; RAW_FILE_NAME=VT_170706_074; RAW_FILE_NAME=VT_170706_076; RAW_FILE_NAME=VT_170706_023 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_09_Plasma Description:POTR_09 Batch=1; RAW_FILE_NAME=VT_170706_079; RAW_FILE_NAME=VT_170706_081; RAW_FILE_NAME=VT_170706_083; RAW_FILE_NAME=VT_170706_080; RAW_FILE_NAME=VT_170706_082; RAW_FILE_NAME=VT_170706_024 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_10_Plasma Description:POTR_10 Batch=1; RAW_FILE_NAME=VT_170706_085; RAW_FILE_NAME=VT_170706_087; RAW_FILE_NAME=VT_170706_089; RAW_FILE_NAME=VT_170706_086; RAW_FILE_NAME=VT_170706_088; RAW_FILE_NAME=VT_170706_025 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_11_Plasma Description:POTR_11 Batch=1; RAW_FILE_NAME=VT_170706_091; RAW_FILE_NAME=VT_170706_093; RAW_FILE_NAME=VT_170706_095; RAW_FILE_NAME=VT_170706_092; RAW_FILE_NAME=VT_170706_094; RAW_FILE_NAME=VT_170706_026 SUBJECT_SAMPLE_FACTORS Study_Sample POTR_12_Plasma Description:POTR_12 Batch=1; RAW_FILE_NAME=VT_170706_097; RAW_FILE_NAME=VT_170706_099; RAW_FILE_NAME=VT_170706_101; RAW_FILE_NAME=VT_170706_098; RAW_FILE_NAME=VT_170706_100; RAW_FILE_NAME=VT_170706_027 SUBJECT_SAMPLE_FACTORS QC q3June2014_1c Description:Q-Standard plasma pool Batch=1; RAW_FILE_NAME=VT_170706_103; RAW_FILE_NAME=VT_170706_105; RAW_FILE_NAME=VT_170706_107; RAW_FILE_NAME=VT_170706_104; RAW_FILE_NAME=VT_170706_106; RAW_FILE_NAME=VT_170706_028 SUBJECT_SAMPLE_FACTORS QC q3June2014_1d Description:Q-Standard plasma pool Batch=1; RAW_FILE_NAME=VT_170706_109; RAW_FILE_NAME=VT_170706_111; RAW_FILE_NAME=VT_170706_113; RAW_FILE_NAME=VT_170706_110; RAW_FILE_NAME=VT_170706_112; RAW_FILE_NAME=VT_170706_029 SUBJECT_SAMPLE_FACTORS QC chearplasma_1c Description:CHEAR plasma pool Batch=1; RAW_FILE_NAME=VT_170706_115; RAW_FILE_NAME=VT_170706_117; RAW_FILE_NAME=VT_170706_119; RAW_FILE_NAME=VT_170706_116; RAW_FILE_NAME=VT_170706_118; RAW_FILE_NAME=VT_170706_030 SUBJECT_SAMPLE_FACTORS QC chearplasma_1d Description:CHEAR plasma pool Batch=1; RAW_FILE_NAME=VT_170706_121; RAW_FILE_NAME=VT_170706_123; RAW_FILE_NAME=VT_170706_125; RAW_FILE_NAME=VT_170706_122; RAW_FILE_NAME=VT_170706_124; RAW_FILE_NAME=VT_170706_031 SUBJECT_SAMPLE_FACTORS QC NIST_1958_2 Description:NIST 1958 Batch=1; RAW_FILE_NAME=VT_170706_127; RAW_FILE_NAME=VT_170706_129; RAW_FILE_NAME=VT_170706_131; RAW_FILE_NAME=VT_170706_128; RAW_FILE_NAME=VT_170706_130; RAW_FILE_NAME=VT_170706_032 #COLLECTION CO:COLLECTION_SUMMARY Eleven adolescents 12–20 years of age undergoing bariatric surgery at CO:COLLECTION_SUMMARY Cincinnati Children’s Hospital between 2006 and 2012 were offered enrollment CO:COLLECTION_SUMMARY in a prospective biospecimen repository protocol (Pediatric Obesity Tissue CO:COLLECTION_SUMMARY Repository [POTR]). Sample recruitment and other POTR features have been CO:COLLECTION_SUMMARY reported previously (Davidson et al. 2017). Intraoperatively, visceral adipose CO:COLLECTION_SUMMARY tissue (vAT) samples from the omentum, abdominal subcutaneous AT (sAT), and CO:COLLECTION_SUMMARY liver samples were obtained by the surgeon and processed immediately in an area CO:COLLECTION_SUMMARY adjacent to the operating room. All samples were snap-frozen in liquid nitrogen, CO:COLLECTION_SUMMARY then stored at −80°C. Plasma was collected pre-operatively after overnight CO:COLLECTION_SUMMARY fasting and stored at -80°C. Written informed consent was obtained from CO:COLLECTION_SUMMARY participants equal to or above 18 years old or from the parent or guardian if CO:COLLECTION_SUMMARY participants were less than 18 years old. The study was approved by the CO:COLLECTION_SUMMARY Institutional Review Board at Cincinnati Children’s Hospital. CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The objective of the observational study was to evaluate the relationship TR:TREATMENT_SUMMARY between adipose and liver tissue POPs and the plasma metabolome. All TR:TREATMENT_SUMMARY participants underwent bariatric surgery at the time of tissue collection. No TR:TREATMENT_SUMMARY other treatment or intervention was evaluated. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples are prepared for metabolomics analysis using established methods SP:SAMPLEPREP_SUMMARY (Johnson et al. (2010). Analyst; Go et al. (2015). Tox Sci). Prior to analysis, SP:SAMPLEPREP_SUMMARY plasma aliquots were removed from storage at -80°C and thawed on ice. Each SP:SAMPLEPREP_SUMMARY cryotube is then vortexed briefly to ensure homogeneity, and 50 μL transferred SP:SAMPLEPREP_SUMMARY to a clean microfuge tube. Immediately after, the plasma is treated with 100 μL SP:SAMPLEPREP_SUMMARY of ice-cold LC-MS grade acetonitrile (Sigma Aldrich) containing 2.5 μL of SP:SAMPLEPREP_SUMMARY internal standard solution with eight stable isotopic chemicals selected to SP:SAMPLEPREP_SUMMARY cover a range of chemical properties. Following addition of acetonitrile, plasma SP:SAMPLEPREP_SUMMARY is then equilibrated for 30 min on ice, upon which precipitated proteins are SP:SAMPLEPREP_SUMMARY removed by centrifuge (16.1 ×g at 4°C for 10 min). The resulting supernatant SP:SAMPLEPREP_SUMMARY (100 μL) is removed, added to a low volume autosampler vial and maintained at SP:SAMPLEPREP_SUMMARY 4°C until analysis (<22 h). SP:SAMPLEPREP_PROTOCOL_ID EmoryUniversity_HRM_SP_082016_01.pdf SP:SAMPLEPREP_PROTOCOL_FILENAME EmoryUniversity_HRM_SP_082016_01.pdf SP:PROCESSING_STORAGE_CONDITIONS Room temperature #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The HILIC column is operated parallel to reverse phase column for simultaneous CH:CHROMATOGRAPHY_SUMMARY analytical separation and column flushing through the use of a dual head HPLC CH:CHROMATOGRAPHY_SUMMARY pump equipped with 10- port and 6-port switching valves. During operation of CH:CHROMATOGRAPHY_SUMMARY HILIC separation method, the MS is operated in positive ion mode and 10 μL of CH:CHROMATOGRAPHY_SUMMARY sample is injected onto the HILIC column while the reverse phase column is CH:CHROMATOGRAPHY_SUMMARY flushing with wash solution. Flow rate is maintained at 0.35 mL/min until 1.5 CH:CHROMATOGRAPHY_SUMMARY min, increased to 0.4 mL/min at 4 min and held for 1 min. Solvent A is 100% CH:CHROMATOGRAPHY_SUMMARY LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is CH:CHROMATOGRAPHY_SUMMARY 2% formic acid (v/v) in LC-MS grade water. Initial mobile phase conditions are CH:CHROMATOGRAPHY_SUMMARY 22.5% A, 75% B, 2.5% C hold for 1.5 min, with linear gradient to 77.5% A, 20% B, CH:CHROMATOGRAPHY_SUMMARY 2.5% C at 4 min, hold for 1 min, resulting in a total analytical run time of 5 CH:CHROMATOGRAPHY_SUMMARY min. During the flushing phase (reverse phase analytical separation), the HILIC CH:CHROMATOGRAPHY_SUMMARY column is equilibrated with a wash solution of 77.5% A, 20% B, 2.5% C. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters XBridge BEH Amide XP HILIC (2.1mm x 50mm x 2.5µm) CH:FLOW_GRADIENT Flow rate is maintained at 0.35 mL/min until 1.5 min, increased to 0.4 mL/min at CH:FLOW_GRADIENT 4 min and held for 1 min CH:FLOW_RATE 0.35- 0.4 mL/min CH:COLUMN_TEMPERATURE 60C CH:INTERNAL_STANDARD [13C6]-D-glucose, [15N,13C5]- L-methionine, [13C5]-L-glutamic acid, CH:INTERNAL_STANDARD [15N]-L-tyrosine, [3,3-13C2]-cystine, [trimethyl- 13C3]-caffeine, [U-13C5, CH:INTERNAL_STANDARD U-15N2]-L-glutamine CH:SAMPLE_INJECTION 10 uL CH:ANALYTICAL_TIME 5 min #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Clinical Biomarkers Laboratory AN:OPERATOR_NAME Vilinh Tran AN:ACQUISITION_DATE July 2017 AN:DATA_FORMAT .Raw #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 high-resolution mass spectrometer was operated at 120,000 resolution and MS:MS_COMMENTS mass-to-charge ratio (m/z) range 85-1275. Probe temperature, capillary MS:MS_COMMENTS temperature, sweep gas and S-Lens RF levels were maintained at 200°C, 300°C, 1 MS:MS_COMMENTS arbitrary units (AU), and 45, respectively. Additional source settings were MS:MS_COMMENTS optimized for sensitivity using a standard mixture, tune settings for sheath MS:MS_COMMENTS gas, auxiliary gas, sweep gas and spray voltage setting were 45 AU, 25 AU and MS:MS_COMMENTS 3.5 kV, respectively. Maximum C-trap injection times were set at 100 MS:MS_COMMENTS milliseconds and automatic gain control target 1 × 106. During untargeted data MS:MS_COMMENTS acquisition, no exclusion or inclusion masses were selected, and data was MS:MS_COMMENTS acquired in MS1 mode only. Raw data files were then extracted using apLCMS (Yu MS:MS_COMMENTS et al. 2009) at five different peak detection settings that have been separately MS:MS_COMMENTS optimized for detection of a wide range of peak intensities and abundances. MS:MS_COMMENTS Peaks detected during each injection were aligned using a mass tolerance of 5 MS:MS_COMMENTS ppm (parts-per-million) and retention grouping was accomplished using MS:MS_COMMENTS non-parametric density estimation grouping, with a maximum retention time MS:MS_COMMENTS deviation of 30 seconds. The resulting feature tables were merged using MS:MS_COMMENTS xMSanalyzer, which identifies overlapping or unique features detected across the MS:MS_COMMENTS different peak detection parameters, and retains the peak with the lowest MS:MS_COMMENTS replicate CV and non-detects for inclusion in the final feature table (Uppal et MS:MS_COMMENTS al. 2013). All R-scripts for data extraction with apLCMS and data merging with MS:MS_COMMENTS xMSanalyzer are provided in the supplementary material. Uniquely detected ions MS:MS_COMMENTS consisted of m/z, retention time and ion abundance, referred to as m/z features. MS:MS_COMMENTS Prior to data analysis, triplicate m/z features averaged and filtered to remove MS:MS_COMMENTS those with triplicate coefficient of variation (CV) ≥ 100% and non-detected MS:MS_COMMENTS values greater than 10%. MS:CAPILLARY_TEMPERATURE 300C MS:ION_SOURCE_TEMPERATURE 200C MS:ION_SPRAY_VOLTAGE 3.5kV MS:IONIZATION Postive MS:MASS_ACCURACY 5ppm MS:SOURCE_TEMPERATURE 200C MS:MS_RESULTS_FILE ST001407_AN002350_Results.txt UNITS:Peak intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END