#METABOLOMICS WORKBENCH Lu_Group_20220325_061043 DATATRACK_ID:3140 STUDY_ID:ST002130 ANALYSIS_ID:AN003483 PROJECT_ID:PR001349 VERSION 1 CREATED_ON April 6, 2022, 7:17 pm #PROJECT PR:PROJECT_TITLE Discovery and characterization of virulence associated functional metabolites in PR:PROJECT_TITLE Escherichia coli based on functional metabolomics strategy PR:PROJECT_SUMMARY Discovery and characterization of virulence associated functional metabolites in PR:PROJECT_SUMMARY Escherichia coli based on functional metabolomics strategy PR:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University PR:LAST_NAME Lu PR:FIRST_NAME Haitao PR:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China PR:EMAIL haitao.lu@sjtu.edu.cn PR:PHONE 15221478139 #STUDY ST:STUDY_TITLE Discovery and characterization of virulence associated functional metabolites in ST:STUDY_TITLE Escherichia coli based on functional metabolomics strategy（pellets ST:STUDY_TITLE metabolomics-1） ST:STUDY_SUMMARY Bacterial metabolites are substrates of virulence factors of uropathogenic ST:STUDY_SUMMARY Escherichia coli (UPEC), but the mechanism underlying the role of functional ST:STUDY_SUMMARY metabolites in bacterial virulence from the perspective of small molecular ST:STUDY_SUMMARY metabolism is unclear. In the present study, we used a strategy of functional ST:STUDY_SUMMARY metabolomics integrated with bacterial genetics in attempt to decipher the ST:STUDY_SUMMARY mechanism of virulence formation in Escherichia coli (E. coli) from the ST:STUDY_SUMMARY viewpoint of small molecule metabolism. We identified the virulence-associated ST:STUDY_SUMMARY metabolome via analysis of the primary metabolome of the pathogenic UTI89 strain ST:STUDY_SUMMARY and the non-pathogenic MG1655 strain. Then, the iron-mediated virulence ST:STUDY_SUMMARY associated metabolome was identified by an iron fishing strategy. Also, the ST:STUDY_SUMMARY mechanism of siderophores in regulating pathogenicity in different environments ST:STUDY_SUMMARY was explored by investigating the effect of iron on siderophore biosynthesis. ST:STUDY_SUMMARY Finally, by knocking out genes related to siderophore biosynthesis, siderophore ST:STUDY_SUMMARY transport and iron utilization, siderophores dependent iron-regulating virulence ST:STUDY_SUMMARY associated metabolome, including 18 functional metabolites, was identified and ST:STUDY_SUMMARY verified to be involved in the regulation of bacterial virulence. Based on this ST:STUDY_SUMMARY we found that these functional metabolites regulated the virulence of E. coli by ST:STUDY_SUMMARY targeting multiple metabolic pathways in an iron-siderophores dependent manner. ST:STUDY_SUMMARY Moreover, a quantitative proteomics approach was implemented to further ST:STUDY_SUMMARY elucidate the mechanism of functional metabolites and functional proteins in ST:STUDY_SUMMARY modulating bacterial virulence. And our findings demonstrated that functional ST:STUDY_SUMMARY proteins regulated the virulence of E. coli by mediating iron binding, ST:STUDY_SUMMARY iron-siderophore transmembrane transport, and the biosynthesis and expression of ST:STUDY_SUMMARY functional metabolites. Interestingly, we found that functional metabolites ST:STUDY_SUMMARY enhance the virulence of E. coli by specifically modulating the key metabolic ST:STUDY_SUMMARY pathways involved in purine metabolism, proline metabolism, arginine metabolism ST:STUDY_SUMMARY and pyrimidine metabolism. Taken together, our study identified for the first ST:STUDY_SUMMARY time 18 functional metabolites regulating the of E. coli virulence, greatly ST:STUDY_SUMMARY enriching our understanding of the mechanism of functional metabolites that ST:STUDY_SUMMARY regulate the E. coli virulence by targeting primary metabolism, which will ST:STUDY_SUMMARY largely contribute to the development of new strategies to target ST:STUDY_SUMMARY virulence-based diagnosis and therapy of infections caused by different ST:STUDY_SUMMARY pathogens. ST:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University ST:LAST_NAME Lu ST:FIRST_NAME Haitao ST:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China ST:EMAIL haitao.lu@sjtu.edu.cn ST:PHONE 15221478139 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Escherichia coli SU:TAXONOMY_ID 562 #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 - WT-0-1P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-1P SUBJECT_SAMPLE_FACTORS - WT-0-2P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-2P SUBJECT_SAMPLE_FACTORS - WT-0-3P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-3P SUBJECT_SAMPLE_FACTORS - WT-0-4P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-4P SUBJECT_SAMPLE_FACTORS - WT-0-5P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-5P SUBJECT_SAMPLE_FACTORS - WT-0-6P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-6P SUBJECT_SAMPLE_FACTORS - WT-10-1P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-1P SUBJECT_SAMPLE_FACTORS - WT-10-2P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-2P SUBJECT_SAMPLE_FACTORS - WT-10-3P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-3P SUBJECT_SAMPLE_FACTORS - WT-10-4P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-4P SUBJECT_SAMPLE_FACTORS - WT-10-5P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-5P SUBJECT_SAMPLE_FACTORS - WT-10-6P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-6P SUBJECT_SAMPLE_FACTORS - MG1655-0-1P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-1P SUBJECT_SAMPLE_FACTORS - MG1655-0-2P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-2P SUBJECT_SAMPLE_FACTORS - MG1655-0-3P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-3P SUBJECT_SAMPLE_FACTORS - MG1655-0-4P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-4P SUBJECT_SAMPLE_FACTORS - MG1655-0-5P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-5P SUBJECT_SAMPLE_FACTORS - MG1655-0-6P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-6P SUBJECT_SAMPLE_FACTORS - ybtS-0-1P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-1P SUBJECT_SAMPLE_FACTORS - ybtS-0-2P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-2P SUBJECT_SAMPLE_FACTORS - ybtS-0-3P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-3P SUBJECT_SAMPLE_FACTORS - ybtS-0-4P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-4P SUBJECT_SAMPLE_FACTORS - ybtS-0-5P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-5P SUBJECT_SAMPLE_FACTORS - ybtS-0-6P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-6P SUBJECT_SAMPLE_FACTORS - BS-0-1P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-1P SUBJECT_SAMPLE_FACTORS - BS-0-2P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-2P SUBJECT_SAMPLE_FACTORS - BS-0-3P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-3P SUBJECT_SAMPLE_FACTORS - BS-0-4P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-4P SUBJECT_SAMPLE_FACTORS - BS-0-5P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-5P SUBJECT_SAMPLE_FACTORS - BS-0-6P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-6P SUBJECT_SAMPLE_FACTORS - entB-0-1P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-1P SUBJECT_SAMPLE_FACTORS - entB-0-2P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-2P SUBJECT_SAMPLE_FACTORS - entB-0-3P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-3P SUBJECT_SAMPLE_FACTORS - entB-0-4P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-4P SUBJECT_SAMPLE_FACTORS - entB-0-5P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-5P SUBJECT_SAMPLE_FACTORS - entB-0-6P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-6P SUBJECT_SAMPLE_FACTORS - iroA-0-1P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-1P SUBJECT_SAMPLE_FACTORS - iroA-0-2P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-2P SUBJECT_SAMPLE_FACTORS - iroA-0-3P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-3P SUBJECT_SAMPLE_FACTORS - iroA-0-4P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-4P SUBJECT_SAMPLE_FACTORS - iroA-0-5P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-5P SUBJECT_SAMPLE_FACTORS - iroA-0-6P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-6P SUBJECT_SAMPLE_FACTORS - irp1-0-1P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-1P SUBJECT_SAMPLE_FACTORS - irp1-0-2P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-2P SUBJECT_SAMPLE_FACTORS - irp1-0-3P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-3P SUBJECT_SAMPLE_FACTORS - irp1-0-4P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-4P SUBJECT_SAMPLE_FACTORS - irp1-0-5P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-5P SUBJECT_SAMPLE_FACTORS - irp1-0-6P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-6P #COLLECTION CO:COLLECTION_SUMMARY After 18h of culture, the sample pellet was isolated. The bacterial pellets CO:COLLECTION_SUMMARY harvested from 50 mL of broth culture were mixed with 1.2 mL 80% ice-cold CO:COLLECTION_SUMMARY methanol (added to internal standard 0.001mg/ml 4-chloro-DL-phenylalanine), then CO:COLLECTION_SUMMARY vortexed for 30 s, and placed on dry ice for 30 min. The samples were CO:COLLECTION_SUMMARY centrifuged at 18000 × g for 15 min at 4 °C. The frozen samples were ground CO:COLLECTION_SUMMARY with beads and the homogenates were centrifuged at 18000 × g for 15 min at 4 CO:COLLECTION_SUMMARY °C. The supernatant was mixed with 800μL ice-cold acetonitrile, and then left CO:COLLECTION_SUMMARY to stand for 20 minutes in an ice bath. After centrifugation at 18000 × g 4℃ CO:COLLECTION_SUMMARY for 15 min, the supernatant was removed and filtered through 0.22μm membrane. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY M63 medium (1.36% monopotassium phosphate, 0.2% ammonium sulfate, 0.024% TR:TREATMENT_SUMMARY magnesium sulfate, 0.001% calcium chloride, and 0.0015% nicotinic acid) was used TR:TREATMENT_SUMMARY to form MG1655 and UTI89 mutants. In addition, add ferric chloride solution to TR:TREATMENT_SUMMARY the medium to prepare 10μM iron M63 medium, we cultured the wild UTI89 strain TR:TREATMENT_SUMMARY in the presence of 10μM iron. The E. coli strain was incubated in LB-agar plate TR:TREATMENT_SUMMARY for 12 hours, one colony was isolated to LB broth for further 4 hours TR:TREATMENT_SUMMARY incubation, then diluted the solution into M63 medium at a ratio of 1:100 and TR:TREATMENT_SUMMARY the cultures were incubated for another18 h at 37°C, 200rpm to culture E. coli. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The bacterial pellets harvested from 50 mL of broth culture were mixed with 1.2 SP:SAMPLEPREP_SUMMARY mL 80% ice-cold methanol (added to internal standard 0.001mg/ml SP:SAMPLEPREP_SUMMARY 4-chloro-DL-phenylalanine), then vortexed for 30 s, and placed on dry ice for 30 SP:SAMPLEPREP_SUMMARY min. The samples were centrifuged at 18000 × g for 15 min at 4 °C. The frozen SP:SAMPLEPREP_SUMMARY samples were ground with beads and the homogenates were centrifuged at 18000 × SP:SAMPLEPREP_SUMMARY g for 15 min at 4 °C. The supernatant was mixed with 800μL ice-cold SP:SAMPLEPREP_SUMMARY acetonitrile, and then left to stand for 20 minutes in an ice bath. After SP:SAMPLEPREP_SUMMARY centrifugation at 18000 × g 4℃ for 15 min, the supernatant was removed and SP:SAMPLEPREP_SUMMARY filtered through 0.22μm membrane. For LC/MS based metabolomics analysis, the SP:SAMPLEPREP_SUMMARY dried samples were dissolved in 200μL water and 5μL aliquots were analyzed. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Agilent MassHunter Workstation Data Acquisition Agilent MassHunter MS:MS_COMMENTS QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ) #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples MG1655-0-1P MG1655-0-2P MG1655-0-3P MG1655-0-4P MG1655-0-5P MG1655-0-6P WT-10-1P WT-10-2P WT-10-3P WT-10-4P WT-10-5P WT-10-6P WT-0-1P WT-0-2P WT-0-3P WT-0-4P WT-0-5P WT-0-6P entB-0-1P entB-0-2P entB-0-3P entB-0-4P entB-0-5P entB-0-6P BS-0-1P BS-0-2P BS-0-3P BS-0-4P BS-0-5P BS-0-6P iroA-0-1P iroA-0-2P iroA-0-3P iroA-0-4P iroA-0-5P iroA-0-6P irp1-0-1P irp1-0-2P irp1-0-3P irp1-0-4P irp1-0-5P irp1-0-6P ybtS-0-1P ybtS-0-2P ybtS-0-3P ybtS-0-4P ybtS-0-5P ybtS-0-6P Factors Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions L-Citrulline 8879.503178 8149.967833 10541.94912 10696.60231 9695.74894 9012.463223 405.7760601 660.163064 189.258733 290.5814728 401.3622227 302.0543219 4339.771698 4726.735425 3439.965914 3910.406351 4303.762259 4510.483236 368.7505195 384.4752104 310.9633065 305.6863978 291.5503369 349.5605223 840.2418136 605.391589 669.8005033 821.3929683 718.5951098 602.4549369 711.3584703 679.1679524 540.1324614 564.2267633 581.0920723 606.1860353 525.633603 505.582061 489.253811 589.7984242 505.6457353 524.0071321 1244.909923 1521.355298 1408.325302 1505.986265 1401.006977 1524.587039 L-Proline 243567.2986 248895.3232 267900.8603 456668.3571 253339.4026 234655.1623 77197.42626 74684.86592 37073.002 47597.42011 44727.88334 53335.10881 8316.956364 10216.35526 9707.866888 9292.752402 9043.792124 10116.16084 18121.95776 23746.20522 10250.87435 9441.012144 55847.82282 23081.78894 15932.73219 15857.72673 14821.53574 17120.38611 17885.94598 15091.26692 2279.561452 2238.865697 1890.632612 2030.595291 2117.825311 2063.017317 112245.3 103014.9766 117340.7874 127768.2777 119302.8999 131786.542 1127.124174 1336.295351 1372.430827 1375.343948 1306.066173 1342.361025 L-Glutamate 244719.6682 244125.256 269463.1709 290469.4774 221104.6893 255473.1548 364.0195446 413.8180616 284.3214529 247.9484219 354.2136957 271.4230512 17503.73785 19332.4837 17782.80043 17401.47747 19451.12762 20642.11482 4976.772638 6304.373482 4788.454415 4376.881496 5555.347409 5745.300302 6872.360271 6271.733119 5620.578788 5489.796998 4989.646282 4234.124654 2992.208535 3310.352496 3003.672016 3197.034404 3326.86491 2910.825638 295509.2589 278879.6034 225405.7066 256459.0938 228298.3144 2199739.167 74987.96485 81435.28154 82350.79129 83326.95606 79137.48402 79569.56784 L-Aspartic acid 23800.68494 25905.09413 29291.3107 28099.57089 26646.95561 22789.90199 2281.351684 1889.622598 1346.131563 1816.948965 1580.029118 2058.565294 3179.294498 4426.140551 3501.57253 3167.639636 4174.19784 5231.5845 640.4113531 671.2134205 520.3134658 439.4260305 955.2289876 658.4522266 1348.525412 1088.57666 986.7969175 929.704462 1069.541405 830.9486578 1092.532268 1049.346643 927.0632198 859.1603737 1040.322973 912.1106689 3316.319057 2384.745343 3398.292351 3599.444103 3136.013107 3049.230224 759.020058 850.9931859 879.6853199 870.6579079 838.3184458 768.4951743 Deoxycytidine 69523.18605 52317.5864 66161.48678 67781.89261 68656.71704 55527.5712 4399.027391 3633.928127 3642.563653 439.9967313 3830.268403 3011.260752 475.1333741 511.2890561 492.7468565 570.9369021 608.4013002 587.6597893 196.4923848 251.4111866 220.1688708 237.9124799 182.349258 181.452059 69.48888418 58.72278828 65.65988509 127.8492277 82.63629246 86.11205956 83.77135407 89.01288667 77.20419934 104.6696375 125.4618296 107.2249 1814.484587 1784.299642 1483.683413 2271.96254 1938.073461 1594.263423 5820.847667 6696.854839 6776.509189 6772.043217 6271.172936 6404.549778 Cytidine 434815.0972 367148.8069 427363.8885 495009.9895 510922.8839 436112.7394 152707.3382 13868.88767 100998.3655 123315.7458 127191.3826 109682.6177 4613.971735 4269.069771 3554.50977 4234.775419 3775.151765 4299.7201 2329.882175 2205.168349 1173.247747 1434.540086 1033.281387 1088.747824 1363.457958 1224.773986 3113.042709 4739.483639 4879.389914 5478.440578 1584.792545 1287.061819 1730.341478 1566.647312 1395.382598 1690.424812 7510.67018 7116.945235 7512.721863 8107.605229 7214.630353 7087.921933 22046.94261 25032.64048 27690.10547 24128.65574 26527.76438 26023.0872 Histamine 495740.4815 437082.7491 530346.0727 602929.4428 545249.7964 484193.6049 147390.9914 208450.5275 104101.6011 132351.0943 130590.8028 188407.2076 11196.59979 11457.86061 9110.365499 10184.08186 10915.78648 11771.0894 4726.741469 4371.078626 3871.456869 4069.008921 4168.122083 4295.451751 8970.624108 8343.941843 8800.464885 8982.710458 9607.865204 9533.33717 3335.133465 2919.112455 2821.296404 2723.857742 3077.961982 3198.018137 39345.44762 36771.6314 38671.20327 42695.11265 36874.31935 36170.62189 42052.31896 43304.58934 53733.62357 49206.13941 48969.06826 51357.34677 L-Arginine 104836.573 82071.54453 110389.7028 105113.5185 101529.7951 90289.0196 12200.93032 11569.77804 7917.997123 7803.365724 11118.55606 9292.565133 42087.82334 47045.01544 48754.72345 47150.10069 47149.4242 47163.47658 4843.280182 6031.049165 4669.255448 3542.652166 4322.719897 4008.360434 7004.181715 5204.534936 6527.782948 6722.489934 6931.805589 6501.934522 7007.378137 6353.026369 6959.134639 6375.692504 7986.975477 7564.471009 15881.44168 15509.31464 21778.0666 17107.60619 14412.33164 13598.22982 7173.270274 7477.521569 7119.154933 7796.089994 7175.850806 7580.724046 L-Lysine 19390.78946 17775.23635 19377.12312 21550.60759 21240.37537 19261.63481 14695.73918 15687.20045 12697.95896 11620.18579 14422.25526 14684.76008 69443.19172 70144.0194 60227.86422 63288.24197 69283.22191 74061.16495 8904.384867 10696.73791 7027.878449 11576.68684 6349.885706 8191.653169 8959.02688 6773.467405 6783.039481 9119.159652 9300.008884 7369.77777 18846.08986 18139.08468 17857.48969 16354.51403 19038.61882 18157.16412 13279.08605 12692.67545 13449.97469 14403.99717 16196.33907 12642.28559 22521.87064 23309.18586 24381.37002 24052.77799 23981.09742 24083.14813 Thymine 381581.6695 330565.8964 420026.9934 452987.6776 387648.2154 369532.3063 81933.91917 68833.60091 61976.98155 80431.41688 122164.5201 87198.74679 208529.091 201392.2159 208589.026 180838.2231 190241.0794 181083.8056 97283.27293 83328.28315 88573.9278 98916.17733 101174.594 78012.55694 105232.1865 78021.79076 64184.97062 119664.2828 74947.6533 102699.8386 120242.6937 111528.4805 111704.6586 129670.8816 118452.0269 128022.3942 165866.8981 137088.7949 107575.0295 148782.8295 160310.7982 13912.22984 142210.0719 167322.5236 170172.9943 173451.3117 154954.8179 159383.6933 5-aminosalicylic acid 33628.29442 30547.94649 31140.57497 32258.23707 34135.65241 33313.59922 34702.1951 42143.01605 26006.38389 28751.16405 56740.62719 27691.29925 122168.0008 118520.9682 134758.9609 109104.6399 116718.4789 117296.6442 18125.99103 12747.27009 11489.92434 11368.91938 11288.14933 10880.83719 15481.68813 11596.53349 10125.06929 24680.94604 19859.9266 14365.6246 30223.04363 26576.58607 23657.03381 30749.10362 26796.80891 27017.4289 35397.33669 34890.35573 40247.47543 44046.61701 41777.97115 33684.23292 12570.72066 12062.04296 8809.946128 22553.17533 13365.92532 14830.55845 Adenosine 5'-monophosphate 93681.01488 98274.04269 111334.2782 111220.7614 110908.494 98952.16835 250092.1155 273367.5321 217161.5115 180069.0339 290964.7482 181377.851 585325.7575 548137.149 535419.0901 515883.6296 540899.7203 543651.648 241525.1491 360925.9389 217946.8363 215404.4897 282855.7625 314211.0856 425810.4756 455542.8768 426384.5442 401500.4942 468961.6716 390428.8331 391808.7375 384513.6037 385726.0638 339688.4132 364337.3094 372588.451 1499118.269 1202647.269 1577206.903 1411428.203 1361975.754 1424298.856 52235.82993 63245.54443 60265.81403 58962.17777 54656.16646 61772.0556 Cytosine 400959.7749 396720.1562 530334.5148 582596.2395 518418.9241 493014.5159 147513.2857 207866.6207 104145.3871 140684.5837 195978.8299 132527.7376 25426.81819 25654.48541 23550.68273 24204.54355 26604.55735 26489.2208 4228.459106 3909.915898 3516.248704 3963.231688 4168.122083 4261.320145 6489.970701 6025.709482 6606.209809 7613.790953 8385.156776 7324.374251 4714.615443 4057.831216 4097.4672 3301.368119 3928.834411 4047.275163 53789.89207 54879.79784 61693.84085 54616.63164 58743.2061 58127.61114 46903.40834 54089.05253 51971.44488 52803.50149 48019.97514 49319.54703 Tryptophan 132813.8561 135100.6073 150768.5009 175951.8801 125209.9444 148033.9935 71060.91199 102006.6448 48517.97138 61645.06759 99439.49645 95586.47697 1165311.062 1114019.577 1062513.34 1047514.276 1096046.353 1118585.73 78418.89258 98874.71874 99889.83016 102887.0884 76615.32375 93167.05098 202402.8501 156939.3408 184065.6793 183499.312 172868.8735 131307.8995 409723.5449 298643.2683 523170.4261 455358.5487 543461.8008 478294.2048 165221.0974 214660.7694 226948.1219 227528.7352 210269.8464 197740.1708 119618.2157 200217.2294 136216.702 102151.2286 142385.8332 165625.1633 Adenine 957772.8081 961488.6455 863299.2457 1275283.748 1168391.265 1087876.424 952462.7456 1078700.105 954868.1891 849693.4515 1244346.109 86813.91294 146544.545 109193.0751 91768.64822 172238.8037 120736.9979 80856.61206 279514.5291 295657.9244 269772.8612 240716.1369 227828.4727 242337.2947 210744.8427 144915.1249 186647.9966 242728.942 246010.5943 178570.8489 186970.7524 177316.2789 200449.7132 153579.2523 186771.2136 187448.8182 1042872.635 1011411.426 1122155.072 1256487.25 1073998.286 955683.1589 2101168.361 2222545.954 2331746.854 2139712.928 2029442.621 2319969.502 L-Ornithine 9135.225198 7057.031317 8960.561869 9487.541434 7283.963259 7580.949465 3824.715802 4737.816899 4430.569247 4140.814446 5606.950212 4704.728983 25708.94993 29010.00562 30122.4304 23620.59639 25082.24785 24651.27836 6397.660071 3948.142403 3988.801503 4684.438843 11577.50042 4728.761672 8594.782391 3344.28361 319.9392196 9297.355784 10065.31921 6673.161937 15230.77839 14173.47722 13457.41294 14399.39303 1584.697079 14341.2432 4740.907726 3749.498806 5008.285172 4873.821447 4678.206302 4833.077865 9144.903656 5877.71889 8389.335003 7135.065484 8264.551816 6279.258167 Phenyllactic acid 1037.45648 986.3834892 1116.035 1138.681324 1088.112921 1035.673744 175.6757115 219.0071857 240.1138251 238.8125111 266.1843335 206.5765573 4578.268196 4347.042168 4549.024383 4389.046394 4607.16938 4787.592478 L-Asparagine 2440.136612 2495.589211 2775.848003 2752.057665 2780.686527 2713.164054 797.1158382 851.2914258 725.37241 816.481727 840.7758536 789.5914777 N-Acetyl-L-methionine 2070.603156 1858.343157 1602.782394 2619.686006 1828.686426 2304.061186 556.5149128 455.7868378 385.9041743 380.4551273 421.044932 528.8729495 L-Histidine 1326.08633 1248.393496 1643.487741 1786.838747 1516.065429 1389.374712 8704.899832 7759.527482 7012.711682 7831.956403 9673.656797 9214.72683 L-Methionine 55255.05204 47779.77487 62396.36518 77814.63912 59577.73987 56638.11523 168929.006 152227.4177 144211.2526 175209.8872 137610.88 148116.6789 2-Deoxyguanosine 23250.96134 21900.52483 20996.84775 24157.1554 22959.61765 21853.13005 8139.745738 7763.967366 7860.900762 12998.95665 13633.0521 10848.97514 N-Acetyl-L-cysteine 1787.82454 1461.755086 1720.98856 1665.660136 1853.065613 1594.388918 10648.59584 10139.89608 8756.493729 8758.916632 9725.255559 9452.638668 L-Homocysteine 3682.773451 3515.334985 3953.841054 4607.072885 3888.195188 3863.725661 1670.1202 2101.229452 1638.629249 1393.596943 1746.828846 1596.655526 Inosine-5'-monophosphate 5414.77652 4533.91671 5644.307746 5606.837852 7140.385838 5545.253062 20341.3258 19191.45333 15017.65655 16564.01645 19219.21396 22651.90597 Betaine 551241.3778 492887.1661 610992.6882 690122.9465 578029.7309 532363.3591 173729.02 175338.6938 148400.7947 157676.4808 169810.0482 163012.1179 Putrescine 148496.0218 148722.4665 169709.5132 169527.7531 181520.5394 163103.2697 70741.23742 64035.60752 66395.4259 57718.68208 63460.952 58868.84887 3-Guanidinopropionic acid  57802.7782 59703.14496 61403.15371 71038.24765 63632.19351 62206.1393 21161.28763 23222.68564 22702.39336 23372.89605 23386.24421 23377.08103 2'-Deoxycytidine 5'-monophosphate 4526.509341 4328.217509 4886.849004 5096.489256 4782.127373 3951.191628 24697.47712 26506.95808 26925.19187 22175.78136 24098.5046 25824.69598 S-(5'-Adenosyl)-L-homocysteine 238578.5675 237919.6735 241844.3326 268269.2845 243664.0488 220650.1018 95585.49456 103285.0322 101057.3137 87108.43682 92427.3223 93890.24247 Spermidine 132896.7609 140587.2 145529.9259 157017.5863 149108.0077 140337.2844 366864.1882 451146.8684 300284.3728 330669.0135 356716.9553 357870.3012 Inosine 1103806.122 1346619.684 1205327.006 1417645.826 1284354.187 1222457.6 329318.4656 321432.1673 305732.4451 301864.4424 340306.1469 338999.1001 Agmatine 876853.9203 843558.4316 919250.4521 1086183.428 898321.586 859132.0143 426842.6841 453530.314 345775.9148 363790.1913 393305.367 419850.1858 Uracil 50452.7954 46484.00757 53935.4828 59793.64453 54108.48228 50512.77036 12883.59567 11753.7675 12026.47349 12361.62263 11217.094 11844.39621 2-hydroxy-2-phenylpropanoic acid 1263.764505 1095.153887 1370.050413 1260.206517 1361.838597 1399.052626 3054.834101 3468.154817 2478.041893 2865.794673 2756.262637 2978.533233 Aminoadipic acid 22349.82704 23301.46348 26380.78256 29695.07005 25054.88388 24359.25086 2797.000248 3227.266626 3143.469997 2834.345446 3199.27962 3198.093386 L-Glutamine 17136.78629 17023.79369 17730.49514 19627.76056 20222.51169 19307.23287 70624.86874 67828.2169 59293.36883 63032.67828 70533.05173 75282.29502 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Molecular weight RT (min) m/z 2'-Deoxycytidine 5'-monophosphate 307.2 1.48 308.07 2-Deoxyguanosine 267.1 3.75 268.27 2-hydroxy-2-phenylpropanoic acid 166.062988 8.3 165.05 3-Guanidinopropionic acid  131.13 1.24 132.14 5-aminosalicylic acid 153.14 1.54 154.15 Adenine 135.13 1.5 136 Adenosine 5'-monophosphate 347.22 1.51 348.07 Agmatine 130.191 0.88 131.1 Aminoadipic acid 161.0688078 1.07 162.08 Betaine 117.15 0.95 118.16 Cytidine 243.22 1.3 244 Cytosine 111.1 1.5 112 Deoxycytidine 227.22 1.5 228 Histamine 111.07 1 112 Inosine 268.23 3.25 269.09 Inosine-5'-monophosphate 348.206 1.57 349.06 L-Arginine 174.2 0.96 175.21 L-Asparagine 132.0534921 0.95 133.06 L-Aspartic acid 133.1 1.05 134.11 L-Citrulline 175.19 0.94 176.2 L-Glutamate 147.13 0.9 148 L-Glutamine 146.146 0.88 147.08 L-Histidine 155.15 0.84 156.16 L-Homocysteine 135.18 1.11 136.05 L-Lysine 146.19 0.8 147.2 L-Methionine 149.21 1.5 150 L-Ornithine 132.19 0.78 133.1 L-Proline 115.13 1 116 N-Acetyl-L-cysteine 163.19 3.38 164.2 N-Acetyl-L-methionine 191.25 6.197 192.26 Phenyllactic acid 166.0629942 8.44 165.05 Putrescine 88.07 0.76 89.08 S-(5'-Adenosyl)-L-homocysteine 384.41 2.72 385.1 Spermidine 145.25 0.71 146.17 Thymine 126.11 2.92 127.12 Tryptophan 204.23 5.71 205.1 Uracil 112.09 1.51 113.1 METABOLITES_END #END