#METABOLOMICS WORKBENCH Codreags00_20200331_131305 DATATRACK_ID:1965 STUDY_ID:ST001339 ANALYSIS_ID:AN002233 PROJECT_ID:PR000915 VERSION 1 CREATED_ON April 3, 2020, 11:25 am #PROJECT PR:PROJECT_TITLE Disruption of Redox Balance Enhances the Effects of BRAF-inhibitors in Melanoma PR:PROJECT_TYPE Untargeted-Targeted Metabolomics analysis PR:PROJECT_SUMMARY Melanomas harboring BRAF mutations can be treated with BRAF inhibitors, but PR:PROJECT_SUMMARY tumor recurrence is inevitable. In spite of large-scale attempts, there remains PR:PROJECT_SUMMARY an unmet need to uncover molecular determinants of BRAFi insensitivity and PR:PROJECT_SUMMARY devise actionable combination targets to overcome resistance. Here, using an PR:PROJECT_SUMMARY integrative approach of experimentation, and mathematical flux balance analyses PR:PROJECT_SUMMARY in a panel of BRAF-mutated melanoma cells, we show that elevated antioxidant PR:PROJECT_SUMMARY capacity of melanoma cells is linked to their drug sensitivity. Specifically, we PR:PROJECT_SUMMARY report that drug-insensitive melanoma cells can maintain higher levels of PR:PROJECT_SUMMARY antioxidant metabolites to withstand the lethal effects of drugs. By extending PR:PROJECT_SUMMARY our analysis to other melanoma subtypes in the TCGA, we show that elevated redox PR:PROJECT_SUMMARY capacity could indeed be a general feature of melanoma. Our results suggest that PR:PROJECT_SUMMARY redox vulnerabilities could be exploited for therapeutic benefits and identify PR:PROJECT_SUMMARY unsuspected combination targets to enhance the effects of BRAFi in pan-melanoma. PR:INSTITUTE Vanderbilt University PR:LAST_NAME Codreanu PR:FIRST_NAME Simona PR:ADDRESS 1234 Stevenson Center Lane PR:EMAIL simona.codreanu@vanderbilt.edu PR:PHONE 6158758422 #STUDY ST:STUDY_TITLE Disruption of Redox Balance Enhances the Effects of BRAF-inhibitors in Melanoma ST:STUDY_SUMMARY Specifically, we report that drug-insensitive melanoma cells can maintain higher ST:STUDY_SUMMARY levels of antioxidant metabolites to withstand the lethal effects of drugs. By ST:STUDY_SUMMARY extending our analysis to other melanoma subtypes in the TCGA, we show that ST:STUDY_SUMMARY elevated redox capacity could indeed be a general feature of melanoma. Our ST:STUDY_SUMMARY results suggest that redox vulnerabilities could be exploited for therapeutic ST:STUDY_SUMMARY benefits and identify unsuspected combination targets to enhance the effects of ST:STUDY_SUMMARY BRAFi in pan-melanoma. ST:INSTITUTE Vanderbilt University ST:LAST_NAME Codreanu ST:FIRST_NAME Simona ST:ADDRESS 1234 Stevenson Center Lane ST:EMAIL simona.codreanu@vanderbilt.edu ST:PHONE 6158758422 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 SKMEL5_SC10 SD1a genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD1_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD1b genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD1_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD2a genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD2_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD2b genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD2_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD3a genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD3_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD3b genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD3_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD4a genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD4_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD4b genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD4_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD5a genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD5_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SD5b genotype:resistant | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SD5_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP1a genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP1_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP1b genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP1_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP2a genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP2_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP2b genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP2_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP3a genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP3_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP3b genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP3_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP4a genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP4_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP4b genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP4_T2 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP5a genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP5_T1 SUBJECT_SAMPLE_FACTORS SKMEL5_SC10 SP5b genotype:resistant | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_SP5_T2 SUBJECT_SAMPLE_FACTORS WM88 WD1a genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD1_T1 SUBJECT_SAMPLE_FACTORS WM88 WD1b genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD1_T2 SUBJECT_SAMPLE_FACTORS WM88 WD2a genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD2_T1 SUBJECT_SAMPLE_FACTORS WM88 WD2b genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD2_T2 SUBJECT_SAMPLE_FACTORS WM88 WD3a genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD3_T1 SUBJECT_SAMPLE_FACTORS WM88 WD3b genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD3_T2 SUBJECT_SAMPLE_FACTORS WM88 WD4a genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD4_T1 SUBJECT_SAMPLE_FACTORS WM88 WD4b genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD4_T2 SUBJECT_SAMPLE_FACTORS WM88 WD5a genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD5_T1 SUBJECT_SAMPLE_FACTORS WM88 WD5b genotype:sensitive | treatment:DMSO RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WD5_T2 SUBJECT_SAMPLE_FACTORS WM88 WP1a genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP1_T1 SUBJECT_SAMPLE_FACTORS WM88 WP1b genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP1_T2 SUBJECT_SAMPLE_FACTORS WM88 WP2a genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP2_T1 SUBJECT_SAMPLE_FACTORS WM88 WP2b genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP2_T2 SUBJECT_SAMPLE_FACTORS WM88 WP3a genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP3_T1 SUBJECT_SAMPLE_FACTORS WM88 WP3b genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP3_T2 SUBJECT_SAMPLE_FACTORS WM88 WP4a genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP4_T1 SUBJECT_SAMPLE_FACTORS WM88 WP4b genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP4_T2 SUBJECT_SAMPLE_FACTORS WM88 WP5a genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP5_T1 SUBJECT_SAMPLE_FACTORS WM88 WP5b genotype:sensitive | treatment:PLX4720 RAW_FILE_NAME=SC_20190501_HILICn_PRM_Redox_Qua_WP5_T2 #COLLECTION CO:COLLECTION_SUMMARY Single-cell derived BRAF-mutated SKMEL5 subclones were derived as previously CO:COLLECTION_SUMMARY described (Paudel et al., 2018). BRAF-mutated melanoma cells (SKMEL5, WM88), CO:COLLECTION_SUMMARY including the SKMEL5 subclones, NRAS-mutated melanoma cells (SKMEL2), and CO:COLLECTION_SUMMARY NF1-mutated melanoma cells (MeWO) were grown and cultured in Dulbecco’s CO:COLLECTION_SUMMARY modified Eagle’s medium and Ham’s F-12 media (DMEM:F12, 1:1, Cat. No. CO:COLLECTION_SUMMARY 11330-032). Media were obtained from Gibco (Grand Island, NY), and supplemented CO:COLLECTION_SUMMARY with 10% fetal bovine serum. All cells were cultured in humidified incubators CO:COLLECTION_SUMMARY that were CO2 and temperature (37oC) controlled. Cells were passaged 1–2 times CO:COLLECTION_SUMMARY per week and were maintained as exponentially growing cultures for a maximum of CO:COLLECTION_SUMMARY less than 20 passages. All cells were tested for mycoplasma, and tested CO:COLLECTION_SUMMARY negative. CO:SAMPLE_TYPE Tumor cells #TREATMENT TR:TREATMENT_SUMMARY PLX4720 (Cat. No. S1152) and vemurafenib (Cat. No. S1267) were obtained from TR:TREATMENT_SUMMARY Selleckchem (Houston, TX). Dabrafenib (Cat No. HY-14660), (1S,3R)-RSL3 (Cat No. TR:TREATMENT_SUMMARY HY-100218A), Ferrostatin-1 (FER1, Cat No. HY-100579), Erastin (Cat No. TR:TREATMENT_SUMMARY HY-15763), (E)-Daporinad (FK866) (Cat No. HY-50876) were obtained from MedChem TR:TREATMENT_SUMMARY Express (Monmouth Junction, NJ) and solubilized in dimethyl sulfoxide (DMSO) at TR:TREATMENT_SUMMARY a stock concentration of 10 mM. Powdered L-Buthionine-sulfoximine (BSO) (Product TR:TREATMENT_SUMMARY No. B2515), powdered Diphenyleneiodonium chloride (DPI) (Product No. D2926), and TR:TREATMENT_SUMMARY powdered L-Glutathione reduced (GSH) were obtained from Sigma-Aldrich. BSO, and TR:TREATMENT_SUMMARY GSH was freshly made at a stock concentration of 100 mM in H2O, while DPI was TR:TREATMENT_SUMMARY solubilized in DMSO at a stock concentration of 10mM. CellRoxTM DeepRed Reagent TR:TREATMENT_SUMMARY (Cat No. C10422) for oxidative stress detection was obtained from ThermoFisher TR:TREATMENT_SUMMARY Scientific. Acetonitrile (AcCN) (Cat No. A955-1), Methanol (MeOH) (Cat No. TR:TREATMENT_SUMMARY A456-1) and water (Cat No. W6-1), Optima LC-MS grade, for the mass spectrometry TR:TREATMENT_SUMMARY analysis were obtained from ThermoFisher Scientific. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The combined global untargeted-targeted metabolomic analysis used BRAF-mutated SP:SAMPLEPREP_SUMMARY melanoma cells (WM88, SKMEL5-SC10) treated with either DMSO or 8μM PLX4720 for SP:SAMPLEPREP_SUMMARY 24 hrs. Cell pellet samples were lysed using 400µL ice cold lysis buffer SP:SAMPLEPREP_SUMMARY (1:1:2, ACN:MeOH:Ammonium Bicarbonate 0.1M, pH 8.0, LC-MS grade) and vortexed SP:SAMPLEPREP_SUMMARY well until the cells mixed well with the solvent. Each sample was sonicated SP:SAMPLEPREP_SUMMARY using a probe tip sonicator, 10 pulses at 30% power, cooling down in ice between SP:SAMPLEPREP_SUMMARY samples. A BCA protein assay was used to determine the protein concentration for SP:SAMPLEPREP_SUMMARY each individual sample, and adjusted to a total amount of protein of 200µg SP:SAMPLEPREP_SUMMARY total protein in 200 µL of lysis buffer. Heavy labeled standard molecules, SP:SAMPLEPREP_SUMMARY Phenylalanine-D8 (CDN Isotopes, Quebec, CA), and Biotin-D2(Cambridge Isotope SP:SAMPLEPREP_SUMMARY Laboratories, Inc., MA, USA), were added to each sample to assess sample SP:SAMPLEPREP_SUMMARY handling steps. Samples were subjected to protein precipitation by addition of SP:SAMPLEPREP_SUMMARY 800µL of ice cold methanol (4x by volume), then incubated at -80°C overnight. SP:SAMPLEPREP_SUMMARY Samples were centrifuged at 10,000 rpm for 10 min to eliminate precipitated SP:SAMPLEPREP_SUMMARY proteins and the metabolite containing supernatant was dried in vacuo and stored SP:SAMPLEPREP_SUMMARY at -80°C until LC-MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Vanquish UHPLC binary system and autosampler CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish UHPLC CH:COLUMN_NAME SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS LC-MS/MS raw data were imported, processed, normalized and reviewed using MS:MS_COMMENTS Progenesis QI v.2.1 (Non-linear Dynamics, Newcastle, UK). All FMS, DDA and PRM MS:MS_COMMENTS sample runs were chromatographically aligned against a QC reference run. MS:MS_COMMENTS Following peak picking, unique spectral features (retention time and m/z pairs) MS:MS_COMMENTS were grouped based on adducts and isotopes, and individual features or MS:MS_COMMENTS metabolites were normalized to all features. Further filtering was carried out MS:MS_COMMENTS by removing features or metabolites that had >30% coefficient of variance. MS:MS_RESULTS_FILE ST001339_AN002233_results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples SD1a SD1b SD2a SD2b SD3a SD3b SD4a SD4b SD5a SD5b SP1a SP1b SP2a SP2b SP3a SP3b SP4a SP4b SP5a SP5b WD1a WD1b WD2a WD2b WD3a WD3b WD4a WD4b WD5a WD5b WP1a WP1b WP2a WP2b WP3a WP3b WP4a WP4b WP5a WP5b Factors genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:DMSO genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:resistant | treatment:PLX4720 genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:DMSO genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 genotype:sensitive | treatment:PLX4720 GSH 5851411.918 5530802.412 5503016.457 5885044.666 4827263.066 5258332.548 5215932.411 5322711.546 5799483.103 5893662.328 4381907.873 4922286.555 5226156.631 5229962.816 4530852.577 5187180.156 5831851.647 5822110.152 4113911.041 4615907.52 4649509.14 4348759.696 4824040.457 5332327.83 4701846.348 4648159.873 4639145.07 4684360.9 4659124.706 5101321.287 578753.6588 373335.7617 756195.7937 602384.5801 424778.4879 360789.17 339548.5087 244959.7904 705444.9894 628214.6078 ATP 7091241.937 6963474.72 7384765.507 8280854.888 7972004.316 9113610.529 6490275.4 6864215.84 6334510.258 6944787.805 3297198.332 3664790.694 4238579.837 4702671.504 3596080.005 4132483.964 3669377.274 3943104.227 2521155.788 2848727.769 7101410.15 7650010.797 7815124.955 9126410.693 6122096.546 6845711.348 6118779.166 6903347.274 7013560.834 7541942.03 4428851.111 4666936.502 5039847.604 5621866.669 3055277.556 3470939.845 2827064.481 3286921.47 3917578.014 4077137.515 GSSG 4481284.702 4629450.388 4134250.87 4779594.886 5820268.655 6788864.026 4123614.67 4240758.173 3951085.719 4272389.254 6815505.055 7874337.676 6786867.517 7048063.941 6349353.99 7216860.081 5918973.148 6331029.353 6999961.002 7553541.953 7051437.701 8198985.974 7114443.073 8317235.631 6458206.015 7144099.197 6371435.638 6980594.376 6741669.592 7255997.467 7858641.992 8528744.106 7416748.365 8305151.192 6361490.925 7010043.113 6681225.723 7664320.303 7356207.832 7379082.015 NADH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 998037.4803 779690.0361 1076642.836 938527.3041 1062941.906 916356.4618 1030795.318 909520.8679 1138439.999 921061.7858 293621.9534 199836.5874 221501.5861 184273.2468 164200.6873 135530.9648 205745.119 184262.2366 237492.6306 202417.3922 UTP 1205127.198 1233727.461 1366949.334 1592093.135 1631310.043 1959804.55 1069170.467 1143578.098 1160517.666 1323546.508 347575.2481 405133.6981 450577.4276 512014.3198 394994.37 461689.4978 409120.4254 465652.2171 218813.278 257181.7461 1520427.655 1749919.38 1494251.405 1933591.451 1199874.135 1429534.196 1138411.813 1336291.34 1158141.49 1338778.835 486095.6979 500277.1938 496170.1404 577567.6662 301252.3182 346132.8625 286973.5044 348030.5856 393466.641 415369.1827 Adenosine 174572.4859 191625.0877 154104.06 177834.1251 148877.7809 173450.4476 176673.6392 192970.4187 184315.4693 206673.2799 222532.8397 243274.0669 195995.257 212629.2681 225405.5742 254385.0183 238691.5201 260870.1586 221580.8481 239894.935 87955.3875 98017.43322 97239.03411 106852.0978 109404.3732 114291.8892 106718.8948 115501.0154 103110.3701 112986.6884 81349.43603 86720.80437 60011.17566 68894.79226 90846.62211 97733.03774 89417.783 99080.36527 76780.52064 91485.37942 GDP 29422.08107 34325.11529 36349.04515 46215.96975 49782.91017 61424.08789 28229.67892 31145.48705 36026.60986 44547.41905 13156.51895 17153.82289 15420.66019 21221.78852 16767.34179 19866.57538 20520.73847 23516.7702 9376.057747 10761.55036 37193.78157 50860.39978 32099.90941 45378.87365 37795.73632 45912.89441 33224.07542 46827.33263 37429.23912 47419.51663 19130.36645 21848.52737 14901.52096 21085.55252 13565.85401 17278.9629 15873.1219 20389.14029 17975.46495 20504.03592 NADP 157218.6948 167335.4095 170997.3818 208351.234 195831.7024 228027.6512 133592.4548 144486.1585 162782.6978 184670.0039 111045.5719 136621.0643 134209.3889 142093.3267 108508.1683 123599.2013 135502.0646 164315.9167 94930.42331 118509.8416 147878.4362 173038.3582 151860.5659 192745.547 148474.4006 158202.5145 137252.3711 151541.2243 128657.6395 142580.6493 100651.0061 102970.8149 102521.2438 118678.0409 86141.90407 101662.4416 82442.99358 98804.83233 87335.33243 101288.9105 UDP 167166.2476 164559.3023 182309.1168 205786.669 218968.2224 251722.0878 140363.3985 155890.6662 157623.826 177935.0489 55590.89501 61360.2577 68452.04783 74715.50474 59394.98436 67455.03424 66755.23276 65300.24601 36462.14763 37897.49772 240583.8692 264465.0133 228599.1188 265525.4355 198573.7398 227212.8568 182292.6004 203983.7753 177706.5222 199748.9582 69572.16964 68821.03286 64532.60112 72846.31982 49177.24524 51171.01807 46396.32083 53405.36548 62416.59564 60299.7951 ADP 613726.3111 568724.0784 549924.0959 522890.9452 556947.7358 550889.7691 554950.3168 535891.3188 535718.0864 505357.5197 377789.1598 370482.8211 461531.385 465020.2223 394966.4548 399483.2599 439407.3463 442338.8069 292316.553 297198.1013 687056.9418 659267.8826 688932.6369 662869.4098 678479.5576 659470.6253 653254.4132 617705.0086 695701.3583 657158.0666 423966.3142 433819.0607 400440.9484 402733.3078 355326.4248 351382.7718 342295.6052 339602.0878 426158.1589 413929.5494 GMP 24594.55541 17466.59857 21573.09222 15599.74551 27481.06374 23006.47965 22187.90041 15245.82073 27721.23006 21240.17465 31572.28081 30930.43735 47979.3784 40057.30637 37195.06418 36649.58724 52259.89857 43190.55401 29226.0105 25255.14158 67352.07856 55032.94063 46283.59668 41364.62702 64542.87125 59908.62204 71425.31167 59728.77092 71776.03166 68704.33473 87901.44589 75593.94482 71972.08041 54115.32057 115125.1546 102817.1303 115879.8759 91430.10974 109816.9987 92581.16594 UMP 30813.72085 27400.35075 30174.95955 32332.88571 27737.94759 32358.13665 37362.25472 36058.92324 46411.15094 47698.14387 83511.71322 92870.90768 129068.7416 125300.0134 104628.7192 121631.2948 137867.9567 135700.4084 95718.8772 112847.1055 104637.9477 109369.9601 105429.5611 121151.3529 130612.0042 132546.9637 170083.5748 169872.5626 167944.9025 189117.8922 94956.89856 96812.63115 94585.73958 101297.963 116806.8398 122658.8071 96995.86471 102090.9709 104039.1463 113070.3235 NAD 1473718.313 1397493.586 1384752.962 1438380.059 1363905.795 1468705.676 1382664.38 1321357.107 1537863.114 1392630.406 1072067.053 1124399.955 1219575.101 1145961.717 1035097.751 1138744.468 1305893.263 1224321.489 1025809.676 1167645.928 343138.3191 439463.0259 426353.8904 567093.734 338874.3553 352078.7936 358643.1098 403649.4325 323506.5313 365683.4406 290888.6523 344930.2247 357778.8172 376667.331 266198.6882 309283.8098 208248.583 236686.8176 271434.5199 301687.6775 AMP 16754.49139 9960.580322 18902.16516 19379.66701 23092.76876 26575.19873 23288.22033 15604.28246 25250.33665 24487.28311 29440.16893 36504.87499 44422.24044 44006.44261 38914.16379 46951.58579 64809.64658 58630.94347 28822.45323 34667.64762 27936.92469 31427.61791 28077.9921 29481.2539 40722.0073 42722.19355 42743.18781 42415.67578 38324.1945 42641.81898 25434.06717 25345.71684 6703.142468 6012.447627 33476.96254 40397.18498 36346.23631 42438.41697 27100.91585 27022.52915 L-Serine 139416.9189 143354.4053 146235.34 161166.9504 124811.0062 133995.1731 136106.1076 146438.7926 146724.8589 156471.5974 187562.704 168878.7256 176292.0949 169347.4057 183620.5224 169047.3548 233987.1201 241352.589 181483.8399 162538.7865 81222.36158 77154.32114 106956.4787 102723.1692 74603.64064 67252.95796 83566.8152 81568.46833 101865.7988 94849.20482 45385.30278 42321.15975 62782.21662 50462.84298 36074.85799 39542.81765 35732.29068 38418.26809 38986.25239 39072.7125 L-Cysteine 1962.631998 837.5877951 1184.510275 818.7292589 1066.309486 323.4146188 1849.855804 1039.511776 1216.212892 833.4726721 2738.193386 1680.135337 4578.831083 2463.699105 3469.805242 2503.348922 4345.439278 2256.583951 3493.636306 2694.235791 4989.895057 2474.022836 4822.336717 3094.882943 2019.08485 3041.306097 4898.667317 3484.039901 3929.308028 2667.472445 9835.924537 3117.796542 10511.45905 5032.954323 13485.99595 10965.12641 9926.358895 6323.289283 16786.74821 11876.12045 GTP 201958.2353 178839.3101 235715.0107 273864.3143 368554.5429 412758.4081 178781.2463 177747.9644 199640.1961 207817.061 46638.22322 58411.56004 73186.93111 77927.4414 57839.66091 74485.16204 71478.74236 76480.69165 29420.3587 37413.14952 196148.7869 214287.3274 183214.9683 226864.1969 118018.4511 149921.4378 119369.42 153178.4331 148759.802 179833.3902 91878.36913 92498.96058 105116.2039 125260.4983 47457.68718 58808.0411 54215.16134 62120.3407 72883.00956 73074.26297 L-Cystine 0 0 0 0 0 0 0 0 0 0 8687.889671 14863.86218 689.5394151 4236.689674 2615.467159 6536.920807 211.3428949 221.6411259 2206.710399 5830.595943 0 0 0 0 0 0 0 0 0 0 74139.53671 148703.8633 14111.15127 31217.90099 262165.033 468273.2856 111827.9887 217449.2432 79678.92466 128273.5412 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention time (min) quantified m/z PubChem ID KEGG ID GSH 10.41903333 306.0767669 3353 C00051 ATP 12.4101 505.9888804 3304 C00002 GSSG 13.18013333 611.1458157 3427 C00127 NADH 9.781733333 664.1189874 3306 C00004 UTP 12.81185 482.9617626 3375 C00075 Adenosine 2.4519 266.0896405 3512 C00212 GDP 12.91676667 442.0173781 3337 C00035 NADP 12.83273333 742.0702315 3308 C00006 UDP 12.81185 402.9952922 3317 C00015 ADP 12.4101 426.0228144 3310 C00008 GMP 11.9641 362.0511244 3444 C00144 UMP 10.75643333 323.0287826 3405 C00105 NAD 10.65028333 662.1033859 3305 C00003 AMP 10.2163 346.0559802 3322 C00020 L-Serine 10.00316667 104.0352712 3365 C00065 L-Cysteine 8.141583333 120.0123963 3397 C00097 GTP 13.71546667 521.9839017 3346 C00044 L-Cystine 13.72615 239.0167574 3774 C00491 METABOLITES_END #END