#METABOLOMICS WORKBENCH lgafeira_20221004_223939 DATATRACK_ID:3492 STUDY_ID:ST002428 ANALYSIS_ID:AN003952 PROJECT_ID:PR001562 VERSION 1 CREATED_ON January 5, 2023, 4:30 pm #PROJECT PR:PROJECT_TITLE Mass Spectrometry-based Proteomic and Metabolomic profiling of serum samples for PR:PROJECT_TITLE discovery and validation of Tuberculosis diagnostic biomarker signature PR:PROJECT_TYPE Proteomic and metabolomic study PR:PROJECT_SUMMARY Tuberculosis (TB) is a transmissible disease listed as one of the 10 leading PR:PROJECT_SUMMARY causes of death worldwide (10 million infected in 2019). A swift and precise PR:PROJECT_SUMMARY diagnosis is essential to forestall its transmission, for which is crucial the PR:PROJECT_SUMMARY discovery of effective diagnostic biomarkers. In this study, we aimed to PR:PROJECT_SUMMARY discover molecular biomarkers for the early diagnosis of tuberculosis. Two PR:PROJECT_SUMMARY independent cohorts comprising 29 and 34 subjects were assayed by proteomics, PR:PROJECT_SUMMARY and 49 were included for metabolomic analysis. All subjects were arranged into 3 PR:PROJECT_SUMMARY experimental groups – healthy controls (Controls), Latent TB infection (LTBI) PR:PROJECT_SUMMARY and TB patients. LC-MS/MS blood serum protein and metabolite levels were PR:PROJECT_SUMMARY submitted to univariate, multivariate and ROC analysis. From the 149 proteins PR:PROJECT_SUMMARY quantified in the discovery set, 25 were found to be differentially abundant PR:PROJECT_SUMMARY between Controls and TB patients. The AUC, specificity and sensitivity, PR:PROJECT_SUMMARY determined by ROC statistical analysis of the model composed by four of these PR:PROJECT_SUMMARY proteins considering both proteomic sets, were 0.96; 93% and 91%, respectively. PR:PROJECT_SUMMARY The five metabolites (9-methyluric acid, indole-3-lactic acid, PR:PROJECT_SUMMARY trans-3-indoleacrylic acid, hexanoylglycine and N-acetyl-L-leucine) that better PR:PROJECT_SUMMARY discriminate the control and TB patient groups (VIP > 1.75) from a total of 92 PR:PROJECT_SUMMARY metabolites quantified in both ionization modes, were submitted to ROC analysis. PR:PROJECT_SUMMARY An AUC=1 was determined with all samples being correctly assigned to the PR:PROJECT_SUMMARY respective experimental group. An integrated ROC analysis enrolling 1 protein PR:PROJECT_SUMMARY and 4 metabolites was also performed for the common control and TB patients in PR:PROJECT_SUMMARY the proteomic and metabolomic groups. This combined signature has correctly PR:PROJECT_SUMMARY assigned the 12 controls and 12 patients used only for prediction (AUC=1, PR:PROJECT_SUMMARY specificity=100% and sensitivity=100%). This multi-omics approach has revealed a PR:PROJECT_SUMMARY biomarker signature for tuberculosis diagnosis that could be potentially used PR:PROJECT_SUMMARY for developing a point-of-care diagnosis clinical test. PR:INSTITUTE ITQB NOVA PR:LABORATORY Proteomics of Non-Model Organisms PR:LAST_NAME Gonçalves PR:FIRST_NAME Luís PR:ADDRESS Avenida Republica PR:EMAIL lgafeira@itqb.unl.pt PR:PHONE 214469464 #STUDY ST:STUDY_TITLE Mass Spectrometry-based Proteomic and Metabolomic profiling of serum samples for ST:STUDY_TITLE discovery and validation of Tuberculosis diagnostic biomarker signature ST:STUDY_SUMMARY Tuberculosis (TB) is a transmissible disease listed as one of the 10 leading ST:STUDY_SUMMARY causes of death worldwide (10 million infected in 2019). A swift and precise ST:STUDY_SUMMARY diagnosis is essential to forestall its transmission, for which is crucial the ST:STUDY_SUMMARY discovery of effective diagnostic biomarkers. In this study, we aimed to ST:STUDY_SUMMARY discover molecular biomarkers for the early diagnosis of tuberculosis. Two ST:STUDY_SUMMARY independent cohorts comprising 29 and 34 subjects were assayed by proteomics, ST:STUDY_SUMMARY and 49 were included for metabolomic analysis. All subjects were arranged into 3 ST:STUDY_SUMMARY experimental groups – healthy controls (Controls), Latent TB infection (LTBI) ST:STUDY_SUMMARY and TB patients. LC-MS/MS blood serum protein and metabolite levels were ST:STUDY_SUMMARY submitted to univariate, multivariate and ROC analysis. From the 149 proteins ST:STUDY_SUMMARY quantified in the discovery set, 25 were found to be differentially abundant ST:STUDY_SUMMARY between Controls and TB patients. The AUC, specificity and sensitivity, ST:STUDY_SUMMARY determined by ROC statistical analysis of the model composed by four of these ST:STUDY_SUMMARY proteins considering both proteomic sets, were 0.96; 93% and 91%, respectively. ST:STUDY_SUMMARY The five metabolites (9-methyluric acid, indole-3-lactic acid, ST:STUDY_SUMMARY trans-3-indoleacrylic acid, hexanoylglycine and N-acetyl-L-leucine) that better ST:STUDY_SUMMARY discriminate the control and TB patient groups (VIP > 1.75) from a total of 92 ST:STUDY_SUMMARY metabolites quantified in both ionization modes, were submitted to ROC analysis. ST:STUDY_SUMMARY An AUC=1 was determined with all samples being correctly assigned to the ST:STUDY_SUMMARY respective experimental group. An integrated ROC analysis enrolling 1 protein ST:STUDY_SUMMARY and 4 metabolites was also performed for the common control and TB patients in ST:STUDY_SUMMARY the proteomic and metabolomic groups. This combined signature has correctly ST:STUDY_SUMMARY assigned the 12 controls and 12 patients used only for prediction (AUC=1, ST:STUDY_SUMMARY specificity=100% and sensitivity=100%). This multi-omics approach has revealed a ST:STUDY_SUMMARY biomarker signature for tuberculosis diagnosis that could be potentially used ST:STUDY_SUMMARY for developing a point-of-care diagnosis clinical test. ST:INSTITUTE ITQB NOVA ST:LABORATORY Proteomics of Non-Model Organisms ST:LAST_NAME Gonçalves ST:FIRST_NAME Luís ST:ADDRESS Avenida Republica ST:EMAIL lgafeira@itqb.unl.pt ST:PHONE 214469464 ST:NUM_GROUPS 3 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 18-65 SU:GENDER Male and female SU:HUMAN_EXCLUSION_CRITERIA HIV patients, respiratory infections beside TB, diabetes, chronic renal failure SU:HUMAN_EXCLUSION_CRITERIA history and transplanted. individuals #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 - C4 Label:Controls RAW_FILE_NAME=C4.mzML SUBJECT_SAMPLE_FACTORS - C5 Label:Controls RAW_FILE_NAME=C5.mzML SUBJECT_SAMPLE_FACTORS - C7 Label:Controls RAW_FILE_NAME=C7.mzML SUBJECT_SAMPLE_FACTORS - C8 Label:Controls RAW_FILE_NAME=C8.mzML SUBJECT_SAMPLE_FACTORS - C9 Label:Controls RAW_FILE_NAME=C9.mzML SUBJECT_SAMPLE_FACTORS - C16 Label:Controls RAW_FILE_NAME=C16.mzML SUBJECT_SAMPLE_FACTORS - C21 Label:Controls RAW_FILE_NAME=C21.mzML SUBJECT_SAMPLE_FACTORS - C25 Label:Controls RAW_FILE_NAME=C25.mzML SUBJECT_SAMPLE_FACTORS - C31 Label:Controls RAW_FILE_NAME=C31.mzML SUBJECT_SAMPLE_FACTORS - C37 Label:Controls RAW_FILE_NAME=C37.mzML SUBJECT_SAMPLE_FACTORS - C38 Label:Controls RAW_FILE_NAME=C38.mzML SUBJECT_SAMPLE_FACTORS - C40 Label:Controls RAW_FILE_NAME=C40.mzML SUBJECT_SAMPLE_FACTORS - C45 Label:Controls RAW_FILE_NAME=C45.mzML SUBJECT_SAMPLE_FACTORS - C46 Label:Controls RAW_FILE_NAME=C46.mzML SUBJECT_SAMPLE_FACTORS - C47 Label:Controls RAW_FILE_NAME=C47.mzML SUBJECT_SAMPLE_FACTORS - C49 Label:Controls RAW_FILE_NAME=C49.mzML SUBJECT_SAMPLE_FACTORS - C51 Label:Controls RAW_FILE_NAME=C51.mzML SUBJECT_SAMPLE_FACTORS - C53 Label:Controls RAW_FILE_NAME=C53.mzML SUBJECT_SAMPLE_FACTORS - C28 Label:LTBI RAW_FILE_NAME=C28.mzML SUBJECT_SAMPLE_FACTORS - C30 Label:LTBI RAW_FILE_NAME=C30.mzML SUBJECT_SAMPLE_FACTORS - CE54 Label:LTBI RAW_FILE_NAME=CE54.mzML SUBJECT_SAMPLE_FACTORS - CE55 Label:LTBI RAW_FILE_NAME=CE55.mzML SUBJECT_SAMPLE_FACTORS - CE57 Label:LTBI RAW_FILE_NAME=CE57.mzML SUBJECT_SAMPLE_FACTORS - DE55 Label:LTBI RAW_FILE_NAME=DE55.mzML SUBJECT_SAMPLE_FACTORS - D10 Label:TB RAW_FILE_NAME=D10.mzML SUBJECT_SAMPLE_FACTORS - D11 Label:TB RAW_FILE_NAME=D11.mzML SUBJECT_SAMPLE_FACTORS - D12 Label:TB RAW_FILE_NAME=D12.mzML SUBJECT_SAMPLE_FACTORS - D13 Label:TB RAW_FILE_NAME=D13.mzML SUBJECT_SAMPLE_FACTORS - D15 Label:TB RAW_FILE_NAME=D15.mzML SUBJECT_SAMPLE_FACTORS - D16 Label:TB RAW_FILE_NAME=D16.mzML SUBJECT_SAMPLE_FACTORS - D19 Label:TB RAW_FILE_NAME=D19.mzML SUBJECT_SAMPLE_FACTORS - D20 Label:TB RAW_FILE_NAME=D20.mzML SUBJECT_SAMPLE_FACTORS - D21 Label:TB RAW_FILE_NAME=D21.mzML SUBJECT_SAMPLE_FACTORS - D22 Label:TB RAW_FILE_NAME=D22.mzML SUBJECT_SAMPLE_FACTORS - D23 Label:TB RAW_FILE_NAME=D23.mzML SUBJECT_SAMPLE_FACTORS - D25 Label:TB RAW_FILE_NAME=D25.mzML SUBJECT_SAMPLE_FACTORS - D26 Label:TB RAW_FILE_NAME=D26.mzML SUBJECT_SAMPLE_FACTORS - D27 Label:TB RAW_FILE_NAME=D27.mzML SUBJECT_SAMPLE_FACTORS - D28 Label:TB RAW_FILE_NAME=D28.mzML SUBJECT_SAMPLE_FACTORS - D56 Label:TB RAW_FILE_NAME=D56.mzML SUBJECT_SAMPLE_FACTORS - D58 Label:TB RAW_FILE_NAME=D58.mzML SUBJECT_SAMPLE_FACTORS - D59 Label:TB RAW_FILE_NAME=D59.mzML SUBJECT_SAMPLE_FACTORS - DE5 Label:TB RAW_FILE_NAME=DE5.mzML SUBJECT_SAMPLE_FACTORS - DE10 Label:TB RAW_FILE_NAME=DE10.mzML SUBJECT_SAMPLE_FACTORS - DE11 Label:TB RAW_FILE_NAME=DE11.mzML SUBJECT_SAMPLE_FACTORS - DE12 Label:TB RAW_FILE_NAME=DE12.mzML SUBJECT_SAMPLE_FACTORS - DE51 Label:TB RAW_FILE_NAME=DE51.mzML SUBJECT_SAMPLE_FACTORS - DE52 Label:TB RAW_FILE_NAME=DE52.mzML SUBJECT_SAMPLE_FACTORS - DE54 Label:TB RAW_FILE_NAME=DE54.mzML SUBJECT_SAMPLE_FACTORS - DE53 Label:TB RAW_FILE_NAME=DE53.mzML #COLLECTION CO:COLLECTION_SUMMARY Peripheral blood samples were collected at the Vendas Novas and Almada-Seixal CO:COLLECTION_SUMMARY Pneumonologic Diagnostic Centers (CDP-). Briefly, around 7 mL of blood was CO:COLLECTION_SUMMARY collected in order to obtain 3 mL of serum. Whole blood samples were harvested CO:COLLECTION_SUMMARY using Clot Activator Tubes (Monovette Serum Gel Z – 7.5 mL, S-monovette, CO:COLLECTION_SUMMARY Sarstedt®). IGRA test (QuantiFERON®-TB Gold IT, ©QIAGEN) was used to confirm CO:COLLECTION_SUMMARY infection status in Control and Latent groups. These samples were transported at CO:COLLECTION_SUMMARY 4°C to the National Institute of Health Doctor Ricardo Jorge (INSA). Samples CO:COLLECTION_SUMMARY displaying hemolysis or with unidentified IGRA results were excluded from CO:COLLECTION_SUMMARY further analysis. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY Blood was allowed to clot for three hours at 4°C after collection. The blood TR:TREATMENT_SUMMARY was then centrifuged at 1000 xg at 4°C for 30 min. Serum collected after TR:TREATMENT_SUMMARY centrifugation was passed through 0.2 μm filters (Sterile Acrodisc®, syringe TR:TREATMENT_SUMMARY filters with Supor membrane, 32 mm) to remove bacteria. Finally, an TR:TREATMENT_SUMMARY anti-protease cocktail (Protease Inhibitor Cocktail, ©SIGMA) was added to the TR:TREATMENT_SUMMARY filtered serum. The samples were transported from INSA to ITQB in a liquid TR:TREATMENT_SUMMARY nitrogen container and stored at −80°C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Serum samples (100 µL) were extracted with 300 µL of methanol overnight at SP:SAMPLEPREP_SUMMARY 4°C. The morning after, the samples were centrifuged for 5 min at 16.000 ×g SP:SAMPLEPREP_SUMMARY and 4°C. The supernatant was transferred to a new tube, evaporated in the SP:SAMPLEPREP_SUMMARY speedvac, and the pellet was stored at −20°C until further analysis. Previous SP:SAMPLEPREP_SUMMARY to LC-MS/MS analysis the pellets were resuspended in 99.16 µL of H2O 0.1%FA. SP:SAMPLEPREP_SUMMARY The Internal Standard (IS) – 3-Nitro-L-tyrosine – was added to a final SP:SAMPLEPREP_SUMMARY concentration of 42 µM. In addition, four pools of samples were used for SP:SAMPLEPREP_SUMMARY metabolite identification. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Dionex UltiMate 3000 UHPLC CH:COLUMN_NAME Waters XBridge C18 (50 x 2.1mm, 3um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Full-MS scan spectra were acquired in the m/z range 75 – 1125, at a resolution MS:MS_COMMENTS of 70,000 (full width at half maximum (FWHM) at m/z 200) and 1×106 automatic MS:MS_COMMENTS gain control (AGC). MS/MS scan spectra were acquired at 17,500 resolution (FWHM MS:MS_COMMENTS at m/z 200), with 1×105 AGC, maximum injection time of 100 ms and dynamic MS:MS_COMMENTS exclusion of 6 s. Raw data files were independently processed by Compound MS:MS_COMMENTS Discoverer™ 3.1 (ThermoFisher Scientific) software for metabolomics data MS:MS_COMMENTS analysis. The preferred database used for metabolite identification was mzCloud MS:MS_COMMENTS – since the "Search mzCloud '' node searches this database for matching MS:MS_COMMENTS fragmentation spectra (MS2) – followed by ChemSpider. For both databases the MS:MS_COMMENTS mass tolerance that the software used to search for matching mass peaks was set MS:MS_COMMENTS at 3 ppm. In the case of the mzCloud search the parameter "FT Fragment Mass MS:MS_COMMENTS Tolerance"was set to 5 ppm. The Human Metabolome Database (HMDB) was selected as MS:MS_COMMENTS the primary source for the ChemSpider search. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ua MS_METABOLITE_DATA_START Samples C4 C5 C7 C8 C9 C16 C21 C25 C31 C37 C38 C40 C45 C46 C47 C49 C51 C53 C28 C30 CE54 CE55 CE57 DE55 D10 D11 D12 D13 D15 D16 D19 D20 D21 D22 D23 D25 D26 D27 D28 D56 D58 D59 DE5 DE10 DE11 DE12 DE51 DE52 DE54 DE53 Factors Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:Controls Label:LTBI Label:LTBI Label:LTBI Label:LTBI Label:LTBI Label:LTBI Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB Label:TB (+/-)12(13)-DiHOME 0.003736250292 0.007811285869 0.00329438603 0.004862703696 0.01780988649 0.004147349051 0.005175085644 0.004455324284 0.007495256397 0.004530157595 0.0053010355 0.00663086728 0.01077353204 0.007934996383 0.007245070155 0.008458383878 0.005518701691 0.01266456275 0.004628787426 0.009391648716 0.009164533334 0.00280449492 0.00626352317 0.005117143557 0.01155193039 0.003870401371 0.01490336354 0.005374081333 0.002866753451 0.007507438124 0.007694572959 0.005215377888 0.04962588764 0.01082509379 0.005935069183 0.01611112079 0.005677160517 0.01248984176 0.02031532236 0.006005192848 0.01915942046 0.01335449834 0.005177223153 0.005975515946 0.00649630351 0.003365919182 0.005854371147 0.007651378028 0.006322159197 0.003900233515 1,3,7-Trimethyluric acid 0.0188216672 0.003387240205 0.002702596127 0.01283270864 0.01528503701 0.00711118384 0.00009849386849 0.01846222442 0.001899707344 0.005053721118 0.008924203399 0.0002398217742 0.01004346611 0.002378112705 0.0003545706954 0.005731410886 0.007125587717 0.008689593169 0.0001319307387 0.00393985883 0.01185840514 0.008509043406 0.006091058071 0.01729081045 0.01010329864 0.000090385035 0.005989678741 0.004939001236 0.00811524829 0.002318900373 0.01963295649 0.005830210659 0.0000799106335 0.0004768204515 0.008868093545 0.0005387748098 0.007710077122 0.0006402245835 0.008146971661 0.00007576258756 0.0000907208752 0.001713335017 0.0002606472375 0.00009184381764 0.00009603116188 0.003723948236 0.006281408541 0.000864264803 0.00009512185577 0.02645501686 1,7-Dimethyluric acid 0.02488676439 0.008448117373 0.009180640137 0.01877513988 0.04819961787 0.008655775083 0.0007451886837 0.02435247204 0.00485013851 0.008348665746 0.01489137883 0.002205266373 0.02128234273 0.004893974967 0.001282128016 0.01569413768 0.009019407989 0.03518235613 0.0009591349767 0.007762443063 0.008974848121 0.01618091766 0.01405481267 0.02093666262 0.007780887051 0.0003571971001 0.008552890989 0.003631092707 0.01299956263 0.006678868155 0.01923844485 0.01605663148 0.0006458047556 0.000595305253 0.01067453258 0.001388613648 0.009413635195 0.003702901859 0.03496470662 0.0001852186541 0.0004569805796 0.004710082104 0.0003485002191 0.0001101242404 0.0003781296629 0.01526174873 0.01760722674 0.003681860894 0.0004262928683 0.09675876143 2-Methylhippuric acid 0.006694581516 0.006559182883 0.01137721637 0.007759544746 0.007758769508 0.01000578447 0.01184886745 0.01276210541 0.008915899955 0.00803749926 0.01384848436 0.01150179223 0.01068685156 0.009439442868 0.005790963925 0.008504842401 0.00708938198 0.00633749508 0.007241632064 0.0103743588 0.01764976142 0.01399448834 0.009408317377 0.008538802797 0.0128736443 0.007197565632 0.005454365843 0.006968757041 0.0064067254 0.009274607216 0.0124815756 0.007874900933 0.01197670202 0.01030050913 0.009438541914 0.0120974268 0.008514658479 0.01140730981 0.01037238958 0.009042552168 0.009541486724 0.01286430804 0.00885082916 0.01000958056 0.01285154503 0.01425929318 0.008926542588 0.008060176513 0.00629719397 0.009602305382 3-Hydroxybutyric acid 0.03430171103 0.07863125844 0.08954629971 0.1097032267 0.1948276547 0.0633043087 0.04065323015 0.05834477627 0.01338590199 0.03095610532 0.08227595998 0.03763499179 0.2198995916 0.04775906723 0.06809144776 0.1395382886 0.01973756328 0.194273577 0.01326168706 0.2447796219 0.07714189081 0.0216669161 0.02500846447 0.01733443162 0.02661454277 0.01463363113 0.1049683125 0.0179232447 0.006073281019 0.04866711615 0.03495545275 0.03031096874 0.5196137435 0.0217340727 0.009056366676 0.0418466548 0.03248275314 0.08066579858 0.0233854624 0.03563855942 0.1586025106 0.312352318 0.02692201076 0.01460505076 0.01068987716 0.008948063308 0.05815032305 0.1080011021 0.06066750174 0.02748715795 3-Indoxyl sulphate 0.1409140713 0.2524963263 0.1640080615 0.2518628105 0.1561574384 0.2264608813 0.235627651 0.3972651006 0.199884195 0.110486705 0.09544142362 0.1325363192 0.225388955 0.1514708347 0.2516459606 0.1317405937 0.1299180309 0.1121983516 0.07075127564 0.148358782 0.3138424037 0.3743809201 0.1590568773 0.1912913379 0.1375203212 0.2115144292 0.05208063215 0.2786789823 0.06402023492 0.1425476265 0.009152916668 0.07901748046 0.08696733969 0.1407435367 0.2672845567 0.2909968598 0.1330542425 0.09864820355 0.1447109558 0.102675806 0.1131335421 0.09285564232 0.4650581989 0.05010870758 0.1618469827 0.2119880291 0.2304993821 0.3890341291 0.7759717876 0.7463658591 3-Methylxanthine 0.05269628772 0.008821771896 0.0351250132 0.05006129879 0.1277989755 0.05320659692 0.004636067406 0.03357933637 0.0227335081 0.02947612903 0.05220939416 0.006460110793 0.03427552833 0.02555184653 0.002369368057 0.04763466062 0.02516503644 0.133741335 0.0034896197 0.01103244764 0.03572173002 0.04236130761 0.06956643419 0.06433942433 0.07897155333 0.0003215872463 0.05347853138 0.0083864451 0.05822434513 0.01343143529 0.1381129104 0.02758506341 0.00106195673 0.001211221717 0.1363634729 0.02543487551 0.04007781819 0.02382268651 0.1255380446 0.001218310398 0.00013311891 0.00914847274 0.002356420854 0.0008712191642 0.001933164064 0.01779631796 0.03901240537 0.0114639926 0.0002889550879 0.1787994942 3-Phenyllactic acid 0.03626446392 0.02324155021 0.04054213307 0.01767451221 0.0204256353 0.05811409787 0.01397824758 0.02555351766 0.01425496471 0.009095300137 0.02388086941 0.0213317926 0.01969844031 0.02210507417 0.01049513295 0.005033278571 0.009471101582 0.01929436955 0.009416838738 0.01499729462 0.01554527047 0.01851898018 0.01999432209 0.0212001431 0.01574331449 0.01287061756 0.02334910655 0.01124477216 0.01023862787 0.011518884 0.01881046506 0.08052940574 0.01159776467 0.01174093017 0.01814358726 0.02129558567 0.03192694555 0.01404578882 0.03618356483 0.01359842155 0.009885376609 0.0085726117 0.01120048213 0.0135160387 0.02329386366 0.02861408322 0.01471485666 0.0281158429 0.02638337351 0.01015973103 4-Acetamidobutanoic acid 0.009800779228 0.01089066545 0.02546036368 0.02217001282 0.01424029802 0.01538739166 0.02485258757 0.02282498588 0.01217582269 0.01830607135 0.02322807533 0.02031348061 0.01842904639 0.01582575359 0.009202111409 0.01687465247 0.00914968536 0.0106802837 0.01016619051 0.01983773554 0.03147995578 0.02516361629 0.01794069277 0.01052742528 0.02135681895 0.01117766837 0.008228149098 0.01436230629 0.01005693708 0.01866816537 0.02294973988 0.01540308091 0.01451364815 0.01685239557 0.01638753687 0.01920224623 0.01578627919 0.01667529223 0.01904193943 0.01474223761 0.02421755884 0.01879229512 0.01127404585 0.01708020498 0.01729615287 0.03166869071 0.01649010273 0.01839799948 0.008626369666 0.01324449305 4-Guanidinobutyric acid 0.006414001192 0.001160568732 0.004211030615 0.001559942753 0.003245709409 0.003416560653 0.008690981366 0.004615983089 0.005059226362 0.001825630283 0.002059402275 0.00266338195 0.001802839834 0.003478592275 0.0009132008972 0.00009180035734 0.0001879975623 0.000162210654 0.004370779136 0.002324749781 0.005218313037 0.004946935586 0.0048367884 0.01072088973 0.001295537612 0.008771063703 0.01173979724 0.006182782152 0.004916411405 0.001691098275 0.006809771628 0.006680171193 0.006356804313 0.002601931861 0.005870730099 0.01029588851 0.007850699746 0.00423004864 0.002934045341 0.007513804802 0.006792532547 0.002877068976 0.009121818561 0.005359806779 0.005186776404 0.004731632141 0.004221666244 0.009529416018 0.0142528795 0.01054027123 4-Hydroxybutyric acid (GHB) 0.2360640354 0.4454403909 0.4851934798 0.5486436793 0.3329073707 0.3337763611 0.3456427855 0.2047473974 0.192788186 0.2034636817 0.5859690182 0.3268548644 0.4594880425 0.2007201004 0.1846689033 0.3220737853 0.1562856913 0.3267983698 0.1687433012 0.4913752448 0.4530576958 0.2843430571 0.2194374241 0.2457017546 0.3559482464 0.123781258 0.554775937 0.2131297045 0.1857050522 0.2695200769 0.3830238833 0.3370418696 0.2890306576 0.1309050252 0.1633705907 0.3169146784 0.3497336263 0.3007099306 0.2916506996 0.2197924867 0.3709510449 0.3820703426 0.137416028 0.3416729044 0.2650360301 0.1942075047 0.1687152999 0.3481655022 0.2813645803 0.2414146796 Capryloylglycine 0.01382552752 0.01381874043 0.004059495022 0.006587149932 0.01239054589 0.003954213663 0.005071680418 0.01573330849 0.01295131132 0.00664799762 0.01057349918 0.007745976492 0.01071657394 0.009452563634 0.005658730233 0.01247350046 0.009189631593 0.005915401152 0.006264712286 0.009433589467 0.0615289086 0.03362917003 0.03867706487 0.07582684131 0.02413799904 0.01152797408 0.01315900297 0.01785622464 0.01638093506 0.02655268716 0.07107076434 0.05656409448 0.02290593742 0.02137829265 0.02207734975 0.05444672483 0.04107260321 0.0726709286 0.06804603106 0.09777686874 0.07802590641 0.02722418595 0.02007667085 0.04534087692 0.02270570847 0.02477312251 0.01316707889 0.01937654374 0.09244201084 0.03941268004 Citric acid 0.2420399439 0.1912668139 0.4231068719 0.2265306714 0.4727161102 0.4242339678 0.3413764523 0.2066176712 0.5437584352 0.1393428687 0.3762042434 0.4321109618 0.43039623 0.1490696535 0.1943994985 0.2393660227 0.2723242591 0.2351830108 0.3554709856 0.7447754965 0.2082003942 0.2224949043 0.1951283209 0.4108458094 0.2225871523 0.113652355 0.212396011 0.2245476641 0.1604344795 0.1767638447 0.1286393041 0.2487546861 0.2264912501 0.178715188 0.2233281321 0.1179380766 0.1366966599 0.1598309341 0.2858488782 0.1078665782 0.2489478899 0.200278042 0.1002038729 0.2039247018 0.1159518366 0.2620288562 0.1092018189 0.1097343726 0.396319999 0.05766644699 D-(+)-Galactose 0.06025827204 0.07071260433 0.1280992027 0.08121551986 0.06759580941 0.1266977982 0.0702864373 0.07335335102 0.0880346986 0.04561260484 0.08106865246 0.08363673883 0.06591573232 0.08116481447 0.05002692517 0.07661346908 0.07716723043 0.08287682662 0.06018288674 0.07976829473 0.08227403856 0.06935404789 0.0577802247 0.06846731677 0.1472722827 0.05621076373 0.155376092 0.05711698297 0.04691771698 0.06735351027 0.06110772352 0.08131714215 0.09141099577 0.07579251288 0.06557229951 0.09955272041 0.1077603753 0.06511424606 0.07752543253 0.07862398549 0.06812498266 0.06483752366 0.05470672672 0.06107626787 0.06358414878 0.06028099779 0.06859649427 0.05689807676 0.05435083248 0.07008251318 DL-4-Hydroxyphenyllactic acid 0.05059214287 0.04933602492 0.04887243416 0.05141505384 0.02708068611 0.04339099866 0.03322371074 0.04089311955 0.02376827649 0.02184267467 0.05280242506 0.0510607252 0.02947332984 0.04140384404 0.02302842914 0.01319329367 0.0168290161 0.03029611753 0.0270097307 0.02503143596 0.04695916243 0.04168663794 0.01998367569 0.03689871873 0.03740285984 0.02774682549 0.02844112938 0.01617547259 0.03399052667 0.02714660899 0.03651873159 0.02458668541 0.0263580525 0.02190688701 0.05063899865 0.02984685532 0.03638453625 0.01695880398 0.04655159763 0.01943343122 0.02190116604 0.02610161328 0.0214600956 0.03707445331 0.02876126918 0.03439540352 0.01855701492 0.03248219545 0.03805539016 0.03570617168 DL-Arginine 0.01083936466 0.01324332657 0.01528505677 0.0122736565 0.01532885096 0.01261906433 0.01287893029 0.009515339865 0.01920665097 0.01034932437 0.01354821595 0.01400190665 0.01151880848 0.01302832398 0.01271236605 0.009632668554 0.009393341095 0.01042390454 0.01409157037 0.009807377624 0.01170636853 0.01429045267 0.01325453885 0.01055996578 0.0164060395 0.01215240216 0.01161796056 0.0117323184 0.01452400695 0.02165521087 0.01264621557 0.01446485767 0.01429482794 0.0128013024 0.01316271687 0.01440391314 0.01559278523 0.01234588273 0.01273450763 0.01221770436 0.009727833917 0.005623129256 0.01847843137 0.01015148459 0.007281838865 0.007646341516 0.009482198372 0.007930384316 0.0172078164 0.008713148915 DL-Tryptophan 0.06780119809 0.07664009483 0.1381922264 0.09273831906 0.07062566801 0.09811071022 0.09317630133 0.08441113981 0.07676833479 0.07225776462 0.09451788388 0.07103440472 0.1098746954 0.08581934815 0.06486780087 0.08341916049 0.06993767874 0.07771516172 0.06534794872 0.07036604943 0.1270466423 0.08428744837 0.08357833249 0.06776125406 0.08484551132 0.0582687754 0.07178494248 0.03335453686 0.04782327835 0.0471109781 0.07258992797 0.0481449363 0.02450855407 0.04098684153 0.04536264752 0.07533482898 0.04729501508 0.07110047933 0.07069229389 0.04502432968 0.04677649494 0.03690353528 0.03982269398 0.05021448403 0.07265767258 0.05297219769 0.04444943184 0.0483151997 0.05845489581 0.04958803253 Glycodeoxycholic acid 0.052359509 0.08561677838 0.0472284068 0.03422383341 0.1570889917 0.1188200876 0.02941395365 0.04200264025 0.1238864828 0.02587121779 0.05389642376 0.04600738833 0.08068626889 0.0357620916 0.05460622433 0.04748528646 0.03777533471 0.1635375333 0.03343007927 0.1692654424 0.04248979131 0.01729254152 0.08820099006 0.1529977452 0.2843893583 0.2114675535 0.2505590126 0.1211340099 0.074746859 0.1747251379 0.2567017635 0.4614823635 0.05186783905 0.2091476793 0.09440745298 0.2237344657 0.4379118363 0.1364196595 0.1728761038 0.02264031038 0.1078164021 0.02108792285 1.091370585 0.1858597724 1.712314575 0.1090434885 0.07226151771 0.1963537702 0.2176202222 0.0311547887 Hexanoylglycine 0.02876490717 0.02476783603 0.003588458736 0.008240778066 0.01753800761 0.004135748565 0.006387054581 0.01589747916 0.01324309109 0.00768081565 0.0141990992 0.009675501127 0.01549170629 0.01443520436 0.008930515301 0.01916393727 0.01414000246 0.006506861375 0.00544755345 0.01209130108 0.1163587047 0.07509492061 0.08486175328 0.1672242361 0.04903157013 0.02197904137 0.02533251985 0.03297767051 0.03335779333 0.05760644883 0.1694232146 0.1245898627 0.0539064815 0.04884643554 0.04613346178 0.1116527386 0.09761923324 0.09191327386 0.1279675217 0.2360842457 0.1598436539 0.06293286122 0.04617132251 0.09869049882 0.05135920441 0.05888128437 0.02094382806 0.04138110159 0.2010123265 0.08595516735 Indole-3-lactic acid 0.04780650056 0.05872636025 0.03948211074 0.05196795382 0.03780661253 0.05363954306 0.05704859377 0.04584632854 0.02405051142 0.03745715139 0.04460318341 0.04924047799 0.05205748737 0.05094203105 0.0324076957 0.02966716049 0.0200398546 0.02747072973 0.01855335707 0.03524326574 0.0434502195 0.05194743325 0.04150532547 0.03779110712 0.0344584117 0.02545036722 0.02146592411 0.0197703181 0.03813096499 0.03039170798 0.05395690022 0.0255123239 0.009356245073 0.01463978611 0.02861153292 0.03270475077 0.03780016897 0.02055164008 0.04219780484 0.02114499054 0.02251952104 0.02138032554 0.02398319927 0.02396671689 0.02428312054 0.03844314081 0.01738654885 0.05378025883 0.0329093094 0.07746590024 Isovanillic acid 0.002472271418 0.001421937514 0.004111320201 0.005479916354 0.007406611813 0.003547185755 0.0005419811995 0.001242214744 0.0003609208586 0.001616485157 0.002803316675 0.00277997324 0.001419020567 0.0007576814018 0.0001841256988 0.0008922616808 0.0007384058389 0.001016616574 0.000840557665 0.001703014834 0.002549050289 0.0005444740103 0.002516446772 0.003823231065 0.001992847935 0.0005330512229 0.001782402493 0.0042181185 0.003961433563 0.003536215441 0.002611056669 0.0009566827075 0.0005302941488 0.0003284110196 0.001655075234 0.000676834534 0.001897786926 0.001734877562 0.002857008537 0.0004750921883 0.0006276025002 0.0004941277927 0.0006572117279 0.0003774395127 0.0004186250369 0.002629874205 0.002455301718 0.002800120511 0.004159202395 0.06778178507 Levulinic acid 0.103943117 0.1694799859 0.152864554 0.2290937921 0.1391185254 0.1696668079 0.1750925919 0.1385771261 0.1289366851 0.07941982638 0.3207968404 0.1179367391 0.1492759626 0.1169841744 0.1035133113 0.1255672147 0.1036547223 0.1591083042 0.07668425647 0.1837812042 0.1702919346 0.1613737783 0.134603327 0.1537775318 0.1201806963 0.08327743504 0.124044454 0.1236028868 0.07091156741 0.1494261543 0.1562365247 0.1541484082 0.1557835752 0.06150195894 0.09480111097 0.2282148466 0.1506718327 0.0912600979 0.2049940207 0.1132397664 0.1604687641 0.1118848416 0.1278336237 0.1264638359 0.10482444 0.08389046372 0.09987553483 0.1155877659 0.143453537 0.06473435829 L-Histidine 0.0330563829 0.03358190931 0.03523075602 0.03973551251 0.02755966136 0.04047340369 0.05131429752 0.04242831314 0.05637792573 0.02464983452 0.03915000725 0.0433039892 0.0533700892 0.0254082712 0.033596212 0.04116956479 0.0418181056 0.04192727278 0.03161426415 0.02688480418 0.03404189416 0.0489151902 0.02232232258 0.03891055804 0.04704715554 0.02268432317 0.02227090425 0.02841801557 0.01315140879 0.03976539336 0.02778798552 0.02850288351 0.009612753313 0.03169630765 0.02049028689 0.02303170824 0.0157222786 0.03176638129 0.03572133559 0.03013074424 0.01975774287 0.02767870847 0.03599980451 0.01936604418 0.01842116046 0.02335943919 0.01539957409 0.01441871589 0.02598193994 0.02055999156 L-Phenylalanine 0.03433079535 0.03929447267 0.06105569221 0.0417507128 0.03646249339 0.05153004333 0.04286595449 0.04220485501 0.04277900129 0.03417535198 0.06450686252 0.04340020927 0.05838663329 0.04685751954 0.03103648224 0.03901624489 0.04078817943 0.05188858982 0.04106266188 0.03892219045 0.05209998712 0.05466327674 0.03985646957 0.04279042918 0.04799826639 0.0315015124 0.04362883129 0.02566368127 0.02477556329 0.03295225162 0.04403275108 0.02593255451 0.04252027194 0.03040767045 0.03310206975 0.05092792866 0.03070327991 0.03849484633 0.03786064548 0.03070511748 0.03259270186 0.03459427176 0.04480678227 0.0353023063 0.03799529422 0.02801114916 0.0336543924 0.03234954956 0.03945688545 0.03323727571 L-Tyrosine 0.06082313632 0.0630099433 0.1282212313 0.07919471179 0.07582821658 0.07082011471 0.0749837805 0.0721393572 0.07048617774 0.05818988208 0.1173778697 0.07392096815 0.07546353711 0.05543925798 0.05257904993 0.03946406591 0.04535554698 0.08505855348 0.06613326311 0.04225896805 0.09133764631 0.06891553427 0.04491262315 0.0763701882 0.08418533517 0.0619869474 0.1049083754 0.03347816141 0.04576257738 0.06672484951 0.0864207456 0.04334206788 0.04722214868 0.05969869241 0.05513751957 0.07483313347 0.05483136875 0.05679629743 0.07312249923 0.03915058632 0.03824957446 0.04454457817 0.06516585586 0.08049790597 0.05452784059 0.03557583255 0.05740621261 0.04500143771 0.06587091237 0.04085538843 N-Isovalerylglycine 0.00938872811 0.01198396736 0.02310815337 0.02043984375 0.01161084317 0.01605948243 0.01952384484 0.01913577365 0.01223219936 0.01664531752 0.02528865843 0.02018506438 0.0211605103 0.01516381937 0.01056428563 0.01489975729 0.01024650552 0.0112944062 0.01024183815 0.02215517776 0.03056737785 0.02268539297 0.01585987982 0.0110475656 0.02351487717 0.01143773386 0.01032025987 0.01180046395 0.01026312396 0.01864583911 0.02411768714 0.01419009278 0.01489542602 0.0170434534 0.01987672689 0.02247438648 0.01504507316 0.02164766296 0.02147510837 0.0147015193 0.01889174586 0.01962175136 0.01204113077 0.01934968895 0.01927294946 0.02925109939 0.01905872168 0.01907334973 0.008196704713 0.01330174227 N-Phenylacetylglutamine 0.0171346525 0.03985226756 0.07943567172 0.02867870691 0.1450845701 0.05778851286 0.04524351426 0.126479541 0.1446270879 0.0318727365 0.0272572652 0.1249998899 0.08587929342 0.1312359404 0.05356515898 0.06573793837 0.06827508953 0.03603967585 0.07356924623 0.04271761216 0.07887828915 0.2056034692 0.01926874724 0.1902397939 0.03297698792 0.01293582597 0.08326732376 0.1195093295 0.09792782999 0.04040989609 0.009038009333 0.06135855159 0.06112231268 0.04046754145 0.05000713766 0.02395697795 0.09156898628 0.03182506393 0.008664671129 0.02327449885 0.07271629465 0.02609869239 0.0634523609 0.02911467636 0.03026356553 0.0266968877 0.07515673711 0.1065524617 0.2740806382 0.7965812007 Pantothenic acid 0.005162351877 0.006142116448 0.009356148876 0.007700881971 0.008106539946 0.006877504107 0.008627122661 0.004500534152 0.005511241613 0.007710264112 0.01106225455 0.008046692108 0.008618370129 0.005785439964 0.003925594666 0.003735121689 0.004636346809 0.004249173161 0.004493942667 0.005976706382 0.005056278586 0.005100731009 0.00547219067 0.004088367131 0.005645386217 0.005023648018 0.004782799671 0.002993826983 0.00375998844 0.00361656611 0.007693615083 0.00829437059 0.003885185579 0.003852131189 0.004211998225 0.01085088193 0.007982837288 0.005850732076 0.005332180868 0.007785263314 0.003368132116 0.006414096495 0.005683550508 0.00406579651 0.004268202874 0.007115137596 0.005677847714 0.007902919892 0.01232916132 0.007054229662 Succinic acid 0.0474624283 0.04759978818 0.07730271358 0.05820253347 0.05882783745 0.06265778069 0.04518082937 0.056251149 0.05314628631 0.04848907677 0.04974205256 0.06270084778 0.05837235759 0.04849795054 0.05027532944 0.05547392141 0.04821115078 0.04423950138 0.03950694715 0.04038133985 0.06760871926 0.0650587857 0.03484751483 0.04992088715 0.05625902537 0.03453163789 0.03981485422 0.04265237086 0.03236186807 0.05094057108 0.06680255568 0.03965610426 0.06153127635 0.05488882157 0.05371517419 0.04940636575 0.05299109224 0.0461761475 0.06950118757 0.04265691314 0.07361354356 0.0542152189 0.04469431494 0.04108123119 0.06798871815 0.05823301516 0.03617887552 0.04103168241 0.040590156 0.04601097316 Uridine 0.05284724747 0.07894576222 0.09226534964 0.1199482513 0.08458679871 0.09388954674 0.08106589263 0.04506346644 0.08951062828 0.07408133407 0.1137306312 0.04481773151 0.070141305 0.08565991179 0.09054901749 0.0705475424 0.07231381714 0.05468783914 0.07773855546 0.08245035132 0.07691364848 0.1005801295 0.09192548644 0.1305163988 0.05748027728 0.05322186079 0.07110166672 0.06985724073 0.06176504014 0.04993374523 0.0825167673 0.06370765697 0.06533906865 0.06847900432 0.0685353231 0.0815899444 0.08636160433 0.09693928095 0.1022319811 0.0667306537 0.1300690189 0.1005437113 0.04103328406 0.07398531725 0.07900901518 0.1001282718 0.07574244565 0.0971321457 0.1193274095 0.08889444809 Vanillin 0.002345629484 0.002619455373 0.005593931323 0.002558419578 0.002018787253 0.001787088439 0.002068653054 0.001457961328 0.002989258516 0.001808141033 0.02398050783 0.0009581973998 0.003568820251 0.002037011594 0.002205741133 0.004836082593 0.005722615107 0.0003914682711 0.003104084665 0.002669405566 0.002246035851 0.002259041901 0.001243239135 0.002174857735 0.001446339173 0.003386303789 0.002211347399 0.002164440713 0.001992486793 0.00155860193 0.003492499387 0.002523284141 0.001657512048 0.001989570677 0.00131870507 0.002292969598 0.002178086876 0.0023228621 0.004187117247 0.007123655333 0.00789422095 0.002325981601 0.02682975032 0.001980318 0.004584243225 0.002374560552 0.00734124488 0.002401514415 0.002513344671 0.005728483389 α-Aspartylphenylalanine 0.001865676314 0.003188374399 0.0009933428008 0.00577977499 0.00238933664 0.002585665899 0.01154009949 0.0088189553 0.006666473707 0.007970706614 0.02007009809 0.03280236284 0.01911555102 0.009585605247 0.005374090671 0.0169009272 0.02205101934 0.01778088751 0.008690734099 0.004729537534 0.01168568513 0.03148998822 0.02107009951 0.01575364853 0.003937666456 0.001601313965 0.001760141544 0.003839709146 0.01262879915 0.03331751031 0.01682056673 0.007971951851 0.02528522747 0.004025659919 0.01443425707 0.01041727421 0.009924665418 0.01712247636 0.005942773384 0.01097351562 0.02257089122 0.03051089122 0.00371244989 0.01222441218 0.004850108437 0.008257175928 0.01104104266 0.005941652574 0.01592555113 0.007128195754 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Molecular Weight RT [min] PubChem KEGG Glycodeoxycholic acid 449.31361 10.353 3035026 C05464 3-Hydroxybutyric acid 104.04724 2.108 441 C01089 DL-4-Hydroxyphenyllactic acid 182.05777 5.884 9378 C03672 3-Indoxyl sulphate 213.00942 6.679 10258 NA Hexanoylglycine 173.10508 6.221 99463 NA Succinic acid 118.0265 2.021 1110 C00042 2-Methylhippuric acid 193.07378 7.184 91637 C01586 Pantothenic acid 219.11043 5.406 6613 C00864 Indole-3-lactic acid 205.07367 7.523 676157 C02043 N-Isovalerylglycine 159.08937 5.508 546304 NA Levulinic acid 116.04719 3.821 11579 NA Capryloylglycine 201.13636 7.462 84290 NA DL-Arginine 174.1114 0.88 232 C02385 4-Guanidinobutyric acid 145.08498 1.193 500 C01035 4-Acetamidobutanoic acid 145.07377 5.732 18189 C02946 D-(+)-Galactose 180.06317 0.906 439357 C00984 N-Phenylacetylglutamine 264.11063 6.513 92258 C04148 Uridine 244.06917 1.183 6029 C00299 Isovanillic acid 168.04214 7.735 12575 NA (+/-)12(13)-DiHOME 314.24547 13.21 53394649 NA 3-Methylxanthine 166.04887 4.549 70639 C16357 3-Phenyllactic acid 166.06282 7.311 3848 C01479 1,7-Dimethyluric acid 196.05945 5.322 91611 C16356 L-Histidine 155.06934 0.88 6274 C00135 4-Hydroxybutyric acid (GHB) 104.04724 2.624 10413 C01991 DL-Tryptophan 204.08977 5.887 9060 C00525 L-Phenylalanine 165.07879 4.823 6140 C00079 1,3,7-Trimethyluric acid 210.07513 5.896 79437 C16361 Citric acid 192.02685 1.524 311 C00158 L-Tyrosine 181.07375 1.964 6057 C00082 α-Aspartylphenylalanine 280.10552 5.964 93078 NA Vanillin 152.04714 7.243 1183 C00755 METABOLITES_END #END