#METABOLOMICS WORKBENCH kaguilera_20210719_133918 DATATRACK_ID:2757 STUDY_ID:ST001902 ANALYSIS_ID:AN003093 PROJECT_ID:PR001197 VERSION 1 CREATED_ON August 11, 2021, 6:00 pm #PROJECT PR:PROJECT_TITLE Metabolomics analysis of AsPC-1 PDAC cells treated with Porcupine inhibitor PR:PROJECT_TITLE (LGK974) PR:PROJECT_SUMMARY WNT signaling promotes pancreatic ductal adenocarcinoma (PDAC) through diverse PR:PROJECT_SUMMARY effects on proliferation, differentiation, survival, and stemness. A subset of PR:PROJECT_SUMMARY PDAC with inactivating mutations in ring finger protein 43 (RNF43) have growth PR:PROJECT_SUMMARY dependency on autocrine WNT ligand signaling, which renders them susceptible to PR:PROJECT_SUMMARY porcupine inhibitors (PORCNi) that block WNT ligand acylation and secretion. For PR:PROJECT_SUMMARY this study, non-targeted metabolomic analyses were performed to explore the PR:PROJECT_SUMMARY therapeutic response of RNF43-mutant PDAC to the PORCNi LGK974. AsPC-1 PR:PROJECT_SUMMARY (RNF43-mutant) PDAC cells were treated with 25 nM LGK974 to explore stable PR:PROJECT_SUMMARY isotope-resolved metabolomics with uniform 1, D-glucose [U13-C6] labeling. PR:INSTITUTE UCLA PR:DEPARTMENT Pathology & Laboratory Medicine PR:LABORATORY Dawson Lab PR:LAST_NAME David PR:FIRST_NAME Dawson PR:ADDRESS 10833 Le Conte Avenue, Los Angeles, CA, 90095, USA PR:EMAIL ddawson@mednet.ucla.edu PR:PHONE (310) 267-2799 #STUDY ST:STUDY_TITLE Metabolomics analysis of AsPC-1 PDAC cells treated with Porcupine inhibitor ST:STUDY_TITLE (LGK974) ST:STUDY_SUMMARY WNT signaling promotes pancreatic ductal adenocarcinoma (PDAC) through diverse ST:STUDY_SUMMARY effects on proliferation, differentiation, survival, and stemness. A subset of ST:STUDY_SUMMARY PDAC with inactivating mutations in ring finger protein 43 (RNF43) have growth ST:STUDY_SUMMARY dependency on autocrine WNT ligand signaling, which renders them susceptible to ST:STUDY_SUMMARY porcupine inhibitors (PORCNi) that block WNT ligand acylation and secretion. For ST:STUDY_SUMMARY this study, non-targeted metabolomic analyses were performed to explore the ST:STUDY_SUMMARY therapeutic response of RNF43-mutant PDAC to the PORCNi LGK974. AsPC-1 ST:STUDY_SUMMARY (RNF43-mutant) PDAC cells were treated with 25 nM LGK974 to explore stable ST:STUDY_SUMMARY isotope-resolved metabolomics with uniform 1, D-glucose [U13-C6] labeling. ST:INSTITUTE University of California, Los Angeles ST:DEPARTMENT Pathology & Laboratory Medicine ST:LABORATORY Dawson Lab ST:LAST_NAME Dawson ST:FIRST_NAME David ST:ADDRESS 10833 LeConte Avenue ST:EMAIL ddawson@mednet.ucla.edu ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 6 ST:PHONE 310-825-0618 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - KA-010 Treatment:Control RAW_FILE_NAME=KA-010 SUBJECT_SAMPLE_FACTORS - KA-011 Treatment:Control RAW_FILE_NAME=KA-011 SUBJECT_SAMPLE_FACTORS - KA-012 Treatment:Control RAW_FILE_NAME=KA-012 SUBJECT_SAMPLE_FACTORS - KA-013 Treatment:25 nM LGK974 RAW_FILE_NAME=KA-013 SUBJECT_SAMPLE_FACTORS - KA-014 Treatment:25 nM LGK974 RAW_FILE_NAME=KA-014 SUBJECT_SAMPLE_FACTORS - KA-016 Treatment:25 nM LGK974 RAW_FILE_NAME=KA-016 #COLLECTION CO:COLLECTION_SUMMARY AsPC-1 PDAC cells were treated with DMSO vehicle control or 25 nM LGK974 (in CO:COLLECTION_SUMMARY triplicate per group). 1 x 10^6 cells were washed with ice-cold 150 mM ammonium CO:COLLECTION_SUMMARY acetate twice before adding 1 mL of ice-cold 80% methanol. After vigorous CO:COLLECTION_SUMMARY vortexing, samples were centrifuged at maximum speed, the aqueous layer was CO:COLLECTION_SUMMARY transferred to a glass vial and the metabolites were dried under vacuum. CO:COLLECTION_SUMMARY Metabolites were resuspended in 50 μL 70% acetonitrile (ACN) and 5 μL of this CO:COLLECTION_SUMMARY solution used for the mass spectrometer-based analysis. The analysis was CO:COLLECTION_SUMMARY performed on a Q Exactive (Thermo Fisher Scientific) in polarity-switching mode CO:COLLECTION_SUMMARY with positive voltage 4.0 kV and negative voltage 4.0 kV. The mass spectrometer CO:COLLECTION_SUMMARY was coupled to an UltiMate 3000RSLC (Thermo Fisher Scientific) UHPLC system. CO:COLLECTION_SUMMARY Mobile phase A was 5 mM ammonium acetate (NH4AcO), pH 9.9, B was acetonitrile CO:COLLECTION_SUMMARY and the separation achieved on a Luna 3 mm NH2 100 A column (150 × 2.0 mm, CO:COLLECTION_SUMMARY Phenomenex). The flow was 200 μL/min, and the gradient ran from 15% A to 95% A CO:COLLECTION_SUMMARY in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 CO:COLLECTION_SUMMARY min. Metabolites were detected and quantified as area under the curve based on CO:COLLECTION_SUMMARY retention time and accurate mass (≤ 3 p.p.m.) using the TraceFinder 3.1 CO:COLLECTION_SUMMARY (Thermo Fisher Scientific). Relative amounts of metabolites between various CO:COLLECTION_SUMMARY conditions, as well as percentage of [13C6] glucose labelling, were calculated CO:COLLECTION_SUMMARY and corrected for naturally occurring 13C abundance. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY AsPC-1 PDAC cells were plated at 500,000 cells per well in 6 well plates in TR:TREATMENT_SUMMARY ultra-low attachment conditions. Cells were pre-treated with 25 nM LGK974 or TR:TREATMENT_SUMMARY DMSO vehicle control for 12 hours, maintained in normal media conditions. Media TR:TREATMENT_SUMMARY was exchanged with [13C6] glucose labeling media and cells were re-treated with TR:TREATMENT_SUMMARY 25 nM LGK974 or DMSO vehicle control for 24 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were washed with ice-cold 150 mM ammonium acetate twice before adding 1 mL SP:SAMPLEPREP_SUMMARY of ice-cold 80% methanol. After vigorous vortexing, samples were centrifuged at SP:SAMPLEPREP_SUMMARY maximum speed, the aqueous layer was transferred to a glass vial and the SP:SAMPLEPREP_SUMMARY metabolites were dried under vacuum. Metabolites were resuspended in 50 μL 70% SP:SAMPLEPREP_SUMMARY acetonitrile (ACN) and 5 μL of this solution used for the mass SP:SAMPLEPREP_SUMMARY spectrometer-based analysis. The analysis was performed on a Q Exactive (Thermo SP:SAMPLEPREP_SUMMARY Fisher Scientific) in polarity-switching mode with positive voltage 4.0 kV and SP:SAMPLEPREP_SUMMARY negative voltage 4.0 kV. The mass spectrometer was coupled to an UltiMate SP:SAMPLEPREP_SUMMARY 3000RSLC (Thermo Fisher Scientific) UHPLC system. Mobile phase A was 5 mM SP:SAMPLEPREP_SUMMARY ammonium acetate (NH4AcO), pH 9.9, B was acetonitrile and the separation SP:SAMPLEPREP_SUMMARY achieved on a Luna 3 mm NH2 100 A column (150 × 2.0 mm, Phenomenex). The flow SP:SAMPLEPREP_SUMMARY was 200 μL/min, and the gradient ran from 15% A to 95% A in 18 min, followed by SP:SAMPLEPREP_SUMMARY an isocratic step for 9 min and re-equilibration for 7 min. Metabolites were SP:SAMPLEPREP_SUMMARY detected and quantified as area under the curve based on retention time and SP:SAMPLEPREP_SUMMARY accurate mass (≤ 3 p.p.m.) using the TraceFinder 3.1 (Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific). Relative amounts of metabolites between various conditions, as well SP:SAMPLEPREP_SUMMARY as percentage of [13C6]glucose labeling, were calculated and corrected for SP:SAMPLEPREP_SUMMARY naturally occurring 13C abundance. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Mobile phase A was 5 mM ammonium acetate (NH4AcO), pH 9.9, B was acetonitrile CH:CHROMATOGRAPHY_SUMMARY and the separation achieved on a Luna 3 mm NH2 100 A column (150 × 2.0 mm, CH:CHROMATOGRAPHY_SUMMARY Phenomenex). The flow was 200 μL/min, and the gradient ran from 15% A to 95% A CH:CHROMATOGRAPHY_SUMMARY in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 CH:CHROMATOGRAPHY_SUMMARY min. CH:CHROMATOGRAPHY_TYPE Normal phase CH:INSTRUMENT_NAME Q Exactive CH:COLUMN_NAME Luna 3 mm NH2 100 A CH:FLOW_RATE 200 uL/min CH:SOLVENT_A 5 mM ammonium acetate (NH4AcO), pH 9.9 CH:SOLVENT_B acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE API MS:ION_MODE POSITIVE MS:MS_COMMENTS Metabolites were detected and quantified as area under the curve based on MS:MS_COMMENTS retention time and accurate mass (≤ 3 p.p.m.) using the TraceFinder 3.1 MS:MS_COMMENTS (Thermo Fisher Scientific). Relative amounts of metabolites between various MS:MS_COMMENTS conditions, as well as percentage of [13C6] glucose labeling, were calculated MS:MS_COMMENTS and corrected for naturally occurring 13C abundance. MS:ION_SPRAY_VOLTAGE 4000 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS area MS_METABOLITE_DATA_START Samples KA-010 KA-011 KA-012 KA-013 KA-014 KA-016 Factors Treatment:Control Treatment:Control Treatment:Control Treatment:25 nM LGK974 Treatment:25 nM LGK974 Treatment:25 nM LGK974 5'-Methyladenosine M0 11773180 11339897 10691357 8359354 2956618 10716900 5'-Methyladenosine M1 519891 158281 NA NA NA NA 5'-Methyladenosine M2 NA 91380 121455 NA NA NA 5'-Methyladenosine M4 528962 784179 234747 NA NA NA 5'-Methyladenosine M5 18583598 22707321 21150185 6125658 2001963 8776714 5'-Methyladenosine M6 NA 463046 460467 NA NA NA Arginine Std 691103577 653104249 641292996 680266568 694412114 636745097 Dimethylglycine M0 137838 204437 209020 265560 344781 397190 Histidine 39178580 45342131 42643521 28427356 26236950 28303851 Histidine Std 493895802 499081565 519688846 491880094 465323914 485130464 Hydroxyproline M0 245984056 261891920 269524749 108822617 99317050 110567715 Hydroxyproline M1 11385503 14263313 14669577 4498946 3459223 4721900 Leucine-Isoleucine Std 3482190488 3509582642 3604417397 3395323570 3298586016 3371759751 Lysine Std 88375442 109539779 105218519 82650520 115854131 55610812 Methionine M0 39672901 48225383 41066621 42182575 37052776 40661377 Methionine M1 289140 2371523 974655 966123 635518 689940 Methionine M4 976103 1076153 551210 363369 NA 312374 Methionine M5 15108252 16599263 15206084 16233303 15538509 16445458 Methionine Std 1052934556 1037485161 1038662733 1026031172 981435897 1009108732 Phenylalanine Std 1709588841 1791193506 1885368524 1719205534 1644418286 1835465493 Pipecolate M0 2326937 2537228 1192978 1626572 968874 830956 Sarcosine M0 10571146 9676130 11808777 3667049 3884631 2525468 Threonine Std 260172272 259340367 275001156 255055061 247731965 239234452 Tyramine 1573021 1008486 2104932 822715 490610 816904 Tyrosine Std 628298855 658720991 696495753 621463565 577592164 638719228 Valine Std 797188955 785265507 810999039 791046236 740825162 790802300 2-Methylpropanoyl-CoA M13 6734 5480 3563 NA NA NA 2-Methylpropanoyl-CoA M18 18127 10925 15355 1840 1969 NA 2-Methylpropanoyl-CoA M19 5051 4615 NA NA NA NA 2-Methylpropanoyl-CoA M2 NA NA NA NA 1847 NA 2-Methylpropanoyl-CoA M4 1627 1560 NA NA NA NA Acetyl-CoA M0 32312 44961 58868 51839 58654 39828 Acetyl-CoA M1 7960 15047 9743 9056 7925 9762 Acetyl-CoA M2 20311 6800 17656 7893 30393 19282 Acetyl-CoA M3 NA NA NA NA 5658 4950 Acetyl-CoA M4 1743 11280 2350 2447 12875 NA Acetyl-CoA M5 56523 59417 83128 35602 50891 28721 Acetyl-CoA M6 7851 12519 30152 1590 10731 2427 Acetyl-CoA M7 22295 34891 24117 11521 15594 9715 Acetyl-CoA M8 NA NA NA NA 2032 NA CoA M0 95486 77781 116965 93031 81850 56497 CoA M1 15071 15908 16975 8918 6348 6962 CoA M2 4344 3114 2916 1678 1968 NA CoA M4 NA 1156 5816 1377 1672 1363 CoA M5 155164 119218 129914 44779 41236 49444 CoA M6 13832 20375 23353 2773 4374 1793 HMG-CoA M5 2902 NA 3862 NA NA NA Propanoyl-CoA M0 NA NA 3442 4109 5385 NA Propanoyl-CoA M1 NA NA NA NA 2316 NA Propanoyl-CoA M19 NA NA 1408 3334 1909 3627 Propanoyl-CoA M5 14424 18068 12130 1935 10201 6797 Propanoyl-CoA M6 2303 NA 10074 NA 4095 1809 Creatine M0 454360570 479535335 483244013 204367381 198196868 210241919 Creatine M1 18158287 18641471 21292485 7701823 7705085 8568217 Creatinine M0 18753089 16791075 15898746 12145876 9801702 9187629 Creatinine M1 425568 88567 210376 NA NA NA GSSG M0 31214217 33720794 34455446 26623537 25030829 24002225 GSSG M1 8305827 8568295 10066783 5859044 6445931 6210901 GSSG M2 13146707 13605570 12310107 7708921 6539089 7964895 GSSG M3 5376521 5831487 5753133 1879667 2418331 4140391 GSSG M4 4643342 5278618 5484159 756004 296998 2789941 GSSG M5 1098471 2531475 2196306 452673 NA 408259 GSSG M6 NA NA 1142492 NA NA NA Nicotinamide 35634926 36106337 35248361 29793120 10960037 28100942 Phosphocreatine M0 28576011 28951976 25779715 11261604 11875693 9174491 Phosphocreatine M1 829919 NA 265380 NA NA NA dA M0 NA NA NA 116609 122131 NA dG M0 60669869 160894703 47395614 62986721 58256593 50441669 dG M1 5360139 16315205 3120181 4098195 4076050 4032374 dG M2 3732089 9970853 1981291 1973424 2090921 1595688 dG M3 1498286 6787325 746777 656646 920642 592019 dG M4 7560191 22454227 4571427 3801795 2890116 2567467 dG M5 104975047 273091445 84682184 46469259 45069190 42091799 dG M6 6573243 18760530 4297528 1912012 1800926 651823 Alanine Std 179345141 185521052 188975893 181729554 868494 174492132 Glycine Std 48226861 48485692 45412175 51076071 41877620 44080749 A M0 58669539 157675039 47679352 62248539 53971094 53066633 A M1 5236595 16309916 4690858 5655142 5457841 4613015 A M2 3879480 10472100 2084109 1873225 2974640 1812840 A M3 1618120 6551879 823277 839177 859265 608534 A M4 8493447 23953329 6221787 3832924 3512497 3270804 A M5 101636377 269572046 85100438 44229150 42207706 42934432 A M6 7087183 17934915 5047052 1790552 1891355 1335599 Adenine M0 805863 928010 1967364 7503268 NA 195021 G M0 1063166 3068109 1155696 1333076 1847536 633578 G M5 NA 4680675 346173 NA 576435 NA Inosine M0 1172639 1041563 378897 1472912 2243268 2378568 Inosine M5 1015767 3751534 1735193 619485 1990435 1067983 U M2 148408 295022 139071 156596 NA 331220 Hypoxanthine M0 1354018 4477090 3309440 2978150 5879226 4060926 Citrulline M0 2343493 1386940 2780360 983798 1584966 2968748 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID Formula m/z RT Polarity 5'-Methyladenosine M0 C00170 C11H15N5O3S 298.0968 3.2 positive 5'-Methyladenosine M1 C00170 C11H15N5O3S 299.1002 3.2 positive 5'-Methyladenosine M2 C00170 C11H15N5O3S 300.1035 3.2 positive 5'-Methyladenosine M4 C00170 C11H15N5O3S 302.1102 3.2 positive 5'-Methyladenosine M5 C00170 C11H15N5O3S 303.1136 3.2 positive 5'-Methyladenosine M6 C00170 C11H15N5O3S 304.1169 3.2 positive Arginine Std C00062 C6H14N4O2 185.1272 9 positive Dimethylglycine M0 C01026 C4H9NO2 104.0706 4.5 positive Histidine C00135 C6H9N3O2 156.0768 8.8 positive Histidine Std C00135 C6H9N3O3 165.0879 8.8 positive Hydroxyproline M0 C01157 C5H9NO3 132.0655 7.2 positive Hydroxyproline M1 C01157 C5H9NO3 133.0689 7.2 positive Leucine-Isoleucine Std C00123 C6H13NO2 139.119 6 positive Lysine Std C00047 C6H14N2O4 155.127 9.5 positive Methionine M0 C00073 C5H11NO2S 150.0583 6.7 positive Methionine M1 C00073 C5H11NO2S 151.0617 6.7 positive Methionine M4 C00073 C5H11NO2S 154.0717 6.7 positive Methionine M5 C00073 C5H11NO2S 155.0751 6.7 positive Methionine Std C00073 C5H11NO2S 156.0721 6.7 positive Phenylalanine Std C00079 C9H11NO3 176.1134 6.5 positive Pipecolate M0 C00408 C6H11NO2 130.0863 9.8 positive Sarcosine M0 C00213 C3H7NO2 90.055 8 positive Threonine Std C00188 C4H9NO3 125.076 8.2 positive Tyramine C00483 C8H11NO 138.0913 2.6 positive Tyrosine Std C00082 C9H11NO3 192.1084 8 positive Valine Std C00183 C5H11NO2 124.1 6.3 positive 2-Methylpropanoyl-CoA M13 C00630 C25H42N7O17P3S 851.2079 22 positive 2-Methylpropanoyl-CoA M18 C00630 C25H42N7O17P3S 856.2247 22 positive 2-Methylpropanoyl-CoA M19 C00630 C25H42N7O17P3S 857.228 22 positive 2-Methylpropanoyl-CoA M2 C00630 C25H42N7O17P3S 840.1711 22 positive 2-Methylpropanoyl-CoA M4 C00630 C25H42N7O17P3S 842.1778 22 positive Acetyl-CoA M0 C00024 C23H38N7O17P3S 810.1331 22 positive Acetyl-CoA M1 C00024 C23H38N7O17P3S 811.1364 22 positive Acetyl-CoA M2 C00024 C23H38N7O17P3S 812.1398 22 positive Acetyl-CoA M3 C00024 C23H38N7O17P3S 813.1431 22 positive Acetyl-CoA M4 C00024 C23H38N7O17P3S 814.1465 22 positive Acetyl-CoA M5 C00024 C23H38N7O17P3S 815.1498 22 positive Acetyl-CoA M6 C00024 C23H38N7O17P3S 816.1532 22 positive Acetyl-CoA M7 C00024 C23H38N7O17P3S 817.1565 22 positive Acetyl-CoA M8 C00024 C23H38N7O17P3S 818.1599 22 positive CoA M0 C00010 C21H36N7O16P3S 768.1225 21.8 positive CoA M1 C00010 C21H36N7O16P3S 769.1258 21.8 positive CoA M2 C00010 C21H36N7O16P3S 770.1292 21.8 positive CoA M4 C00010 C21H36N7O16P3S 772.1359 21.8 positive CoA M5 C00010 C21H36N7O16P3S 773.1392 21.8 positive CoA M6 C00010 C21H36N7O16P3S 774.1426 21.8 positive HMG-CoA M5 C00356 C27H44N7O20P3S 917.1815 22.5 positive Propanoyl-CoA M0 C00100 C24H40N7O17P3S 824.1487 21.5 positive Propanoyl-CoA M1 C00100 C24H40N7O17P3S 825.1521 21.5 positive Propanoyl-CoA M19 C00100 C24H40N7O17P3S 843.2124 21.5 positive Propanoyl-CoA M5 C00100 C24H40N7O17P3S 829.1655 21.5 positive Propanoyl-CoA M6 C00100 C24H40N7O17P3S 830.1688 21.5 positive Creatine M0 C00300 C4H9N3O2 132.0768 7 positive Creatine M1 C00300 C4H9N3O2 133.0801 7 positive Creatinine M0 C00300 C4H7N3O1 114.0662 3.9 positive Creatinine M1 C00300 C4H7N3O1 115.0695 3.9 positive GSSG M0 C00127 C20H32N6O12S2 613.1592 17 positive GSSG M1 C00127 C20H32N6O12S2 614.1626 17 positive GSSG M2 C00127 C20H32N6O12S2 615.1659 17 positive GSSG M3 C00127 C20H32N6O12S2 616.1693 17 positive GSSG M4 C00127 C20H32N6O12S2 617.1726 17 positive GSSG M5 C00127 C20H32N6O12S2 618.176 17 positive GSSG M6 C00127 C20H32N6O12S2 619.1793 17 positive Nicotinamide C00153 C6H6N2O 123.0553 2.8 positive Phosphocreatine M0 C02305 C4H10N3O5P 212.0431 16.1 positive Phosphocreatine M1 C02305 C4H10N3O5P 213.0464 16.1 positive dA M0 C00559 C10H13N5O3 252.1086 3.7 positive dG M0 C00330 C10H13N5O4 268.1035 4 positive dG M1 C00330 C10H13N5O4 269.1069 4 positive dG M2 C00330 C10H13N5O4 270.1102 4 positive dG M3 C00330 C10H13N5O4 271.1136 4 positive dG M4 C00330 C10H13N5O4 272.1169 4 positive dG M5 C00330 C10H13N5O4 273.1203 4 positive dG M6 C00330 C10H13N5O4 274.1236 4 positive Alanine Std C00041 C3H7NO2 94.062 7 positive Glycine Std C00037 C2H5NO2 79.043 8.7 positive A M0 C00212 C10H13N5O4 268.104 4 positive A M1 C00212 C10H13N5O4 269.1074 4 positive A M2 C00212 C10H13N5O4 270.1107 4 positive A M3 C00212 C10H13N5O4 271.1141 4 positive A M4 C00212 C10H13N5O4 272.1174 4 positive A M5 C00212 C10H13N5O4 273.1208 4 positive A M6 C00212 C10H13N5O4 274.1241 4 positive Adenine M0 C00147 C5H5N5 136.0618 5.4 positive G M0 C00387 C10H13N5O5 284.099 7.7 positive G M5 C00387 C10H13N5O5 289.1157 7.7 positive Inosine M0 C00294 C10H12N4O5 269.0881 8.6 positive Inosine M5 C00294 C10H12N4O5 274.1048 8.6 positive U M2 C00299 C9H12N2O6 247.0835 4.9 positive Hypoxanthine M0 C00262 C5H4N4O 137.0458 8.3 positive Citrulline M0 C00327 C6H13N3O3 176.103 8.7 positive METABOLITES_END #END