#METABOLOMICS WORKBENCH nmatulionis_20230907_122351 DATATRACK_ID:4296 STUDY_ID:ST002852 ANALYSIS_ID:AN004673 PROJECT_ID:PR001786 VERSION 1 CREATED_ON September 7, 2023, 1:44 pm #PROJECT PR:PROJECT_TITLE MYC is a regulator of androgen receptor inhibition-induced metabolic PR:PROJECT_TITLE requirements in prostate cancer PR:PROJECT_SUMMARY Advanced prostate cancers are treated with therapies targeting the androgen PR:PROJECT_SUMMARY receptor (AR) signaling pathway. While many tumors initially respond to AR PR:PROJECT_SUMMARY inhibition, nearly all develop resistance. It is critical to understand how PR:PROJECT_SUMMARY prostate tumor cells respond to AR inhibition in order to exploit PR:PROJECT_SUMMARY therapy-induced phenotypes prior to the outgrowth of treatment-resistant PR:PROJECT_SUMMARY disease. Here, we comprehensively characterize the effect of AR blockade on PR:PROJECT_SUMMARY prostate cancer metabolism using transcriptomics, metabolomics and bioenergetics PR:PROJECT_SUMMARY approaches. The metabolic response to AR inhibition is defined by reduced PR:PROJECT_SUMMARY glycolysis, robust elongation of mitochondria, and increased reliance on PR:PROJECT_SUMMARY mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling PR:PROJECT_SUMMARY as mediators of AR blockade-induced metabolic phenotypes. Rescuing DRP1 PR:PROJECT_SUMMARY phosphorylation after AR inhibition restores mitochondrial fission, while PR:PROJECT_SUMMARY rescuing MYC restores glycolytic activity and prevents sensitivity to complex I PR:PROJECT_SUMMARY inhibition. Our study provides new insight into the regulation of PR:PROJECT_SUMMARY treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer. PR:INSTITUTE University of California, Los Angeles PR:DEPARTMENT Biological Chemistry PR:LABORATORY Heather Christofk PR:LAST_NAME Matulionis PR:FIRST_NAME Nedas PR:ADDRESS 615 Charles E Young Dr S, BSRB 354-05 PR:EMAIL nmatulionis@mednet.ucla.edu PR:PHONE 3102060163 #STUDY ST:STUDY_TITLE MYC is a regulator of androgen receptor inhibition-induced metabolic ST:STUDY_TITLE requirements in prostate cancer ST:STUDY_SUMMARY Advanced prostate cancers are treated with therapies targeting the androgen ST:STUDY_SUMMARY receptor (AR) signaling pathway. While many tumors initially respond to AR ST:STUDY_SUMMARY inhibition, nearly all develop resistance. It is critical to understand how ST:STUDY_SUMMARY prostate tumor cells respond to AR inhibition in order to exploit ST:STUDY_SUMMARY therapy-induced phenotypes prior to the outgrowth of treatment-resistant ST:STUDY_SUMMARY disease. Here, we comprehensively characterize the effect of AR blockade on ST:STUDY_SUMMARY prostate cancer metabolism using transcriptomics, metabolomics and bioenergetics ST:STUDY_SUMMARY approaches. The metabolic response to AR inhibition is defined by reduced ST:STUDY_SUMMARY glycolysis, robust elongation of mitochondria, and increased reliance on ST:STUDY_SUMMARY mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling ST:STUDY_SUMMARY as mediators of AR blockade-induced metabolic phenotypes. Rescuing DRP1 ST:STUDY_SUMMARY phosphorylation after AR inhibition restores mitochondrial fission, while ST:STUDY_SUMMARY rescuing MYC restores glycolytic activity and prevents sensitivity to complex I ST:STUDY_SUMMARY inhibition. Our study provides new insight into the regulation of ST:STUDY_SUMMARY treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer. ST:INSTITUTE University of California, Los Angeles ST:DEPARTMENT Biological Chemistry ST:LABORATORY Heather Christofk ST:LAST_NAME Matulionis ST:FIRST_NAME Nedas ST:ADDRESS 615 Charles E Young Dr S, BSRB 354-05 ST:EMAIL nmatulionis@mednet.ucla.edu ST:PHONE 3102060163 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:GENOTYPE_STRAIN NSG #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 - tumor-veh-1-01 Treatment:Vehicle | Technical Replicate (Region of Tumor):1 RAW_FILE_NAME=tumor-veh-1-01.RAW SUBJECT_SAMPLE_FACTORS - tumor-veh-1-02 Treatment:Vehicle | Technical Replicate (Region of Tumor):2 RAW_FILE_NAME=tumor-veh-1-02.RAW SUBJECT_SAMPLE_FACTORS - tumor-veh-2-01 Treatment:Vehicle | Technical Replicate (Region of Tumor):1 RAW_FILE_NAME=tumor-veh-2-01.RAW SUBJECT_SAMPLE_FACTORS - tumor-veh-2-02 Treatment:Vehicle | Technical Replicate (Region of Tumor):2 RAW_FILE_NAME=tumor-veh-2-02.RAW SUBJECT_SAMPLE_FACTORS - tumor-veh-3-01 Treatment:Vehicle | Technical Replicate (Region of Tumor):1 RAW_FILE_NAME=tumor-veh-3-01.RAW SUBJECT_SAMPLE_FACTORS - tumor-veh-3-02 Treatment:Vehicle | Technical Replicate (Region of Tumor):2 RAW_FILE_NAME=tumor-veh-3-02.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-1-01 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 RAW_FILE_NAME=tumor-enzalutamide-1-01.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-1-02 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 RAW_FILE_NAME=tumor-enzalutamide-1-02.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-2-01 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 RAW_FILE_NAME=tumor-enzalutamide-2-01.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-2-02 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 RAW_FILE_NAME=tumor-enzalutamide-2-02.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-3-01 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 RAW_FILE_NAME=tumor-enzalutamide-3-01.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-3-02 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 RAW_FILE_NAME=tumor-enzalutamide-3-02.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-4-01 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 RAW_FILE_NAME=tumor-enzalutamide-4-01.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-4-02 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 RAW_FILE_NAME=tumor-enzalutamide-4-02.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-5-01 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 RAW_FILE_NAME=tumor-enzalutamide-5-01.RAW SUBJECT_SAMPLE_FACTORS - tumor-enzalutamide-5-02 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 RAW_FILE_NAME=tumor-enzalutamide-5-02.RAW #COLLECTION CO:COLLECTION_SUMMARY Tumors were collected after 10 days of treatment and prepared for histology, CO:COLLECTION_SUMMARY protein extraction, and metabolite extraction. CO:SAMPLE_TYPE Prostate #TREATMENT TR:TREATMENT_SUMMARY 7 million 16D cells were implanted subcutaneously with 100 μl Matrigel TR:TREATMENT_SUMMARY (Corning) into NSG mice to form primary tumors. Primary tumors were harvested, TR:TREATMENT_SUMMARY minced, and re-implanted (20 - 80 mg of minced tumor tissue with 100 μl TR:TREATMENT_SUMMARY Matrigel per mouse) into NSG mice. 16D tumor-bearing mice were treated by oral TR:TREATMENT_SUMMARY gavage with 10 mg/kg/day of Enzalutamide in the vehicle (1% carboxymethyl TR:TREATMENT_SUMMARY cellulose, 0.5% Tween 80, and 5% dimethylsulfoxide) or the vehicle only, with a TR:TREATMENT_SUMMARY two-days-on/one-day-off schedule. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After tumor dissection, a maximum of 30mg of tissue was weighed, snap frozen, SP:SAMPLEPREP_SUMMARY and stored at -80C until metabolite extraction. To extract metabolites, SP:SAMPLEPREP_SUMMARY weighed tumor tissue was added to a bead tube (Thermo Fisher Scientific) SP:SAMPLEPREP_SUMMARY containing 1ml 80% methanol plus 10mM potassium trifluoromethanesulfonate (TMSO) SP:SAMPLEPREP_SUMMARY internal standard on ice. Samples were homogenized for 1 minute at max speed on SP:SAMPLEPREP_SUMMARY a bead homogenizer (Thermo Fisher Scientific). Bead tubes were spun at 17000g at SP:SAMPLEPREP_SUMMARY 4C for 10 minutes. The supernatant was transferred to an Eppendorf tube and SP:SAMPLEPREP_SUMMARY spun at 17000g at 4C for 10 minutes. A volume of extraction equivalent to 3mg SP:SAMPLEPREP_SUMMARY of tumor tissue was transferred to an ABC vial (Thermo Fisher Scientific). All SP:SAMPLEPREP_SUMMARY volumes were normalized to 500 µl with 80% methanol containing TMSO internal SP:SAMPLEPREP_SUMMARY standard. 80% MeOH was evaporated from the ABC vials using the EZ-2Elite SP:SAMPLEPREP_SUMMARY evaporator (Genevac) and samples were stored at -80°C until analysis. Dried SP:SAMPLEPREP_SUMMARY metabolites were reconstituted in 100 µL of a 50% acetonitrile (ACN) 50% dH20 SP:SAMPLEPREP_SUMMARY solution. Samples were vortexed and spun down for 10 minutes at 17,000g. 70 µL SP:SAMPLEPREP_SUMMARY of the supernatant was then transferred to HPLC glass vials. 10 µL of these SP:SAMPLEPREP_SUMMARY metabolite solutions were injected per analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were run on a Vanquish (Thermo Fisher Scientific) UHPLC system with CH:CHROMATOGRAPHY_SUMMARY mobile phase A (20mM ammonium carbonate, pH 9.7) and mobile phase B (100% ACN) CH:CHROMATOGRAPHY_SUMMARY at a flow rate of 150 µL/min on a SeQuant ZIC-pHILIC Polymeric column (2.1 × CH:CHROMATOGRAPHY_SUMMARY 150 mm 5 μm, EMD Millipore) at 35°C. Separation was achieved with a linear CH:CHROMATOGRAPHY_SUMMARY gradient from 20% A to 80% A in 20 minutes followed by a linear gradient from CH:CHROMATOGRAPHY_SUMMARY 80% A to 20% A from 20 minutes to 20.5 minutes. 20% A was then held from 20.5 CH:CHROMATOGRAPHY_SUMMARY minutes to 28 minutes. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A 20 mM Ammonium carbonate, pH 9.7 CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT Check Summary CH:FLOW_RATE 150 µL/min CH:COLUMN_TEMPERATURE 35°C #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Heather Christofk AN:OPERATOR_NAME Nedas Matulionis #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The UHPLC was coupled to a Q-Exactive (Thermo Fisher Scientific) mass analyzer MS:MS_COMMENTS running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, MS:MS_COMMENTS aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For MS:MS_COMMENTS both polarities mass scan settings were kept at full-scan-range=(70-1000), MS:MS_COMMENTS ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data MS:MS_COMMENTS was also collected from the top three most abundant singly-charged ions in each MS:MS_COMMENTS scan with normalized-collision-energy=35. Each of the resulting “.RAW” files MS:MS_COMMENTS was then centroided and converted into two “.mzXML” files (one for positive MS:MS_COMMENTS scans and one for negative scans) using msconvert from ProteoWizard12. These MS:MS_COMMENTS “.mzXML” files were imported into the MZmine 2 software package13. Ion MS:MS_COMMENTS chromatograms were generated from MS1 spectra via the built-in Automated Data MS:MS_COMMENTS Analysis Pipeline (ADAP) chromatogram module14 and peaks were detected via the MS:MS_COMMENTS ADAP wavelets algorithm. Peaks were aligned across all samples via the Random MS:MS_COMMENTS sample consensus aligner module, gap-filled, and assigned identities using an MS:MS_COMMENTS exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our MS:MS_COMMENTS in-house MS1-RT database. Peak boundaries and identifications were then further MS:MS_COMMENTS refined by manual curation. Peaks were quantified by area under the curve MS:MS_COMMENTS integration and exported as CSV files. If stable isotope tracing was used in the MS:MS_COMMENTS experiment, the peak areas were additionally processed via the R package MS:MS_COMMENTS AccuCor15 to correct for natural isotope abundance. Peak areas for each sample MS:MS_COMMENTS were normalized by the measured area of the internal standard MS:MS_COMMENTS trifluoromethanesulfonate (present in the extraction buffer) and by the number MS:MS_COMMENTS of cells present in the extracted well. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples tumor-enzalutamide-1-01 tumor-enzalutamide-1-02 tumor-enzalutamide-2-01 tumor-enzalutamide-2-02 tumor-enzalutamide-3-01 tumor-enzalutamide-3-02 tumor-enzalutamide-4-01 tumor-enzalutamide-4-02 tumor-enzalutamide-5-01 tumor-enzalutamide-5-02 tumor-veh-1-01 tumor-veh-1-02 tumor-veh-2-01 tumor-veh-2-02 tumor-veh-3-01 tumor-veh-3-02 Factors Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):1 Treatment:Enzalutamide | Technical Replicate (Region of Tumor):2 Treatment:Vehicle | Technical Replicate (Region of Tumor):1 Treatment:Vehicle | Technical Replicate (Region of Tumor):2 Treatment:Vehicle | Technical Replicate (Region of Tumor):1 Treatment:Vehicle | Technical Replicate (Region of Tumor):2 Treatment:Vehicle | Technical Replicate (Region of Tumor):1 Treatment:Vehicle | Technical Replicate (Region of Tumor):2 6-phosphogluconic acid 308111.5166 117848.5436 379253.3239 283329.8783 348483.689 192986.7785 543540.8082 300493.4954 363515.8575 319568.3619 72852.18814 81437.06909 493910.1523 460077.8792 240179.8158 194895.3325 acetyl-CoA 200976.4626 62730.22175 196015.0967 148830.5216 261294.1215 147586.5086 315259.5062 184623.0867 143294.7937 130512.1224 16786.7952 34508.7972 108782.6342 87892.9961 137618.9638 161979.704 adenylosuccinic acid 1510138.884 974074.8816 2606399.743 1276632.609 2139589.532 1404469.709 3782478.039 1426589.829 1439121.429 1760098.309 172726.0951 247398.991 1945870.924 2643738.624 1292278.549 1817637.048 ADP 2.87E+07 1.31E+07 3.80E+07 2.26E+07 3.82E+07 2.07E+07 4.61E+07 1.95E+07 3.37E+07 5.20E+07 3090473.678 1.07E+07 2.31E+07 3.00E+07 3.21E+07 2.34E+07 AICAR 111076.4213 62145.54366 154526.8657 114191.855 214976.2915 110462.771 162444.9057 104041.5716 100213.6646 143791.611 29175.53689 40622.33742 81255.26743 77449.2905 75682.59948 52208.36103 alpha-ketoglutarate 2753992.998 6766677.272 8961334.463 4466580.3 3213993.456 5467827.864 8824009.353 4695251.716 1.18E+07 1.91E+07 1158918.809 5213567.686 2155849.476 7013570.042 4949538.896 3934983.637 AMP 1.28E+08 9.55E+07 1.79E+08 1.05E+08 1.68E+08 1.16E+08 2.13E+08 1.24E+08 1.13E+08 1.53E+08 2.19E+07 4.29E+07 1.29E+08 1.52E+08 9.83E+07 1.08E+08 arachidic acid 2143847.195 4847444.899 1913445.912 2196367.951 2046252.88 6255850.127 1742266.84 2950982.399 5080524.344 1884953.104 2.05E+07 1.37E+07 3767395.946 4111657.936 4077763.432 1.33E+07 argininosuccinate 637207.072 261459.7193 1099239.615 625754.9332 1391727.439 541138.2445 1251159.891 395544.1426 516659.1747 575483.9721 64164.10008 110534.1525 519562.4292 604433.3827 464111.1468 343112.6161 ascorbate 7.74E+07 5.79E+07 2.62E+08 1.22E+08 1.70E+07 2.46E+07 4.73E+08 2.13E+08 6.25E+07 2.32E+08 2840415.541 773195.7355 1.13E+08 1.19E+08 6.27E+07 1.88E+08 aspartate 7.54E+07 2.93E+07 1.01E+08 7.27E+07 1.05E+08 5.98E+07 9.52E+07 6.30E+07 4.40E+07 6.44E+07 1.48E+07 3.33E+07 1.04E+08 1.13E+08 6.22E+07 8.46E+07 ATP 8097426.054 2072051.648 1.23E+07 6722996.247 9994851.368 4168721.592 1.21E+07 4135837.854 8714758.924 2.58E+07 778222.038 4241516.838 5767760.619 8008835.809 1.32E+07 5723168.932 CAIR 40292.7804 61914.2878 34960.28779 53742.55871 26541.51754 56502.5864 35323.07682 54785.29898 51239.06572 91663.14911 9457.395732 22303.72864 41391.3549 49694.89998 38260.22059 70438.77705 carbamoyl-aspartate 41786.38816 232603.9645 261683.472 142381.3065 50616.60108 85155.81672 214182.5498 212062.2212 100079.2421 142413.0403 0 9471.597301 32054.51609 443086.7165 196330.5478 404152.8253 CDP 1260303.45 695523.5588 1109478.615 867670.0658 790382.5716 543269.535 971816.0932 686514.9349 1002264.942 1692876.024 203645.6245 823971.5568 1007928.779 1222761.477 1103359.513 739643.8521 CDP-ethanolamine 8323032.632 5101002.764 1.40E+07 8498415.746 1.47E+07 7896989.129 1.74E+07 9110634.053 1.02E+07 1.45E+07 1463292.345 3777053.305 8028683.573 1.03E+07 7671924.139 8325448.451 cis-aconitate 3048596.871 3356873.36 5907939.598 3763101.279 4113134.749 4176250.573 4789331.503 4077212.145 3891714.209 6545483.324 2492034.292 4283372.619 2421134.981 5922229.688 2846243.541 5865341.478 citrate / isocitrate 7.26E+07 7.37E+07 1.37E+08 8.57E+07 1.12E+08 9.24E+07 1.29E+08 8.88E+07 7.72E+07 1.90E+08 2.29E+07 7.64E+07 4.52E+07 1.34E+08 7.21E+07 1.33E+08 CMP 5217357.03 4202441.431 4871819.542 3209806.977 4341027.526 3456562.211 3948679.058 3506914.828 3260348.68 4675227.407 1780547.527 2569475.471 5957438.857 7919553.366 2809746.235 3935069.285 CoA 1178135.277 411078.2642 1419061.616 753580.2323 1278167.781 630966.1259 1356864.426 772650.1043 528865.1174 938956.667 55835.24545 108364.3435 827579.4744 854027.4441 742258.8576 729157.136 CTP 333193.6097 90906.50941 452618.2987 303154.0188 251368.6282 131857.4296 269190.5736 144824.6679 224469.5063 772931.717 53153.89538 333061.7895 271649.1354 399022.5138 374880.5199 170359.1065 cysteine 40129.10782 71615.5166 57642.16211 75626.74512 62136.89493 97890.47266 52613.16711 81216.39844 51595.75098 84167.33301 72409.03809 65211.81055 188618.4676 65719.85241 88686.29927 174043.0997 cystine 1394311.287 983999.16 946860.1556 1289672.156 1193692.18 1853470.177 1054175.264 1157818.453 1111717.735 747419.5951 2351361.639 1075159.77 1212193.394 1228397.989 606303.1999 447070.949 cytidine 3202237.631 2962954.947 3095576.356 2178148.726 3150251.822 3353591.587 4094933.853 3248380.089 1668332.89 1550864.14 3455070.267 1150489.634 2518984.371 2920970.191 1785183.468 3862388.409 dCTP 57331.29706 20118.30122 53245.61606 24388.78516 34563.51305 34204.20151 57595.85547 34875.50523 14628.55322 45378.21902 13540.93897 58034.25802 85298.49838 9043.575195 70843.86397 53162.14279 dihydroorotate 0 110595.228 65357.98619 25008.82509 13638.75815 20625.71823 32190.21077 57214.54856 35020.87333 39934.22135 0 27811.87371 11613.11861 82020.34079 116454.5056 273422.6763 dihydroxyacetone-phosphate 1707458.185 897893.2263 1915310.72 2163309.762 1982101.479 2190250.244 2013794.826 3735270.242 1950314.255 1490741.172 803386.8619 430365.2049 2957432.429 2296350.695 1672300.81 2021117.024 dTMP 1253443.159 837959.1899 921457.5545 1123206.993 708538.8964 868053.1845 1057357.078 1331432.937 918548.2214 505443.1979 1060409.732 838927.9514 2386273.916 1820710.583 1022861.586 1914980.576 dTTP 68435.34968 0 26972.11281 74049.57556 0 0 0 0 18979.3209 23946.25059 21916.125 0 73780.84414 114475.1827 108751.4646 66580.05093 FGAR 0 70994.4478 0 28069.92075 54574.81234 58714.29699 20890.02043 65052.36501 23388.01505 47588.2765 0 30475.31363 18692.89015 244463.6442 26965.20496 68211.30495 fructose 2562414.371 2688229 3679847.578 2926615.87 3819804.93 3316779.9 1.39E+07 1.16E+07 9284253.934 1.15E+07 1257285.872 2126483.81 2992443.838 5946820.843 3050376.386 3801529.777 fructose-1;6-bisphosphate 5318986.724 1541776.38 8191872.45 5133105.362 8658973.424 3253106.29 9544285.917 5762513.372 4153135.603 5480272.142 382537.6524 787185.5399 6658192.932 5338804.609 3314438.699 2810377.074 fructose-1-phosphate 30402.05859 50952.18066 329430.7645 215461.3899 353541.9121 199241.1831 493331.776 232176.8721 273168.221 201062.1135 761.1254883 22382.79883 71910.53027 223377.298 201893.8289 183801.828 fructose-6-phosphate 5453241.964 1452029.332 1.19E+07 6401543.675 1.34E+07 4159583.305 2.18E+07 9398208.087 6312170.268 5868958.574 918906.271 892589.3606 8770866.092 5545923.346 3135826.82 2383438.928 fumarate 2.44E+07 1.57E+07 3.62E+07 2.11E+07 2.93E+07 2.23E+07 4.42E+07 2.40E+07 2.83E+07 2.45E+07 7843177.612 7579668.312 2.61E+07 2.89E+07 2.69E+07 2.62E+07 gamma-amino-butyrate 1.08E+07 8651412.775 1.33E+07 8981725.851 1.24E+07 1.05E+07 1.64E+07 1.09E+07 1.24E+07 1.43E+07 3381415.404 5910164.115 1.06E+07 1.30E+07 9045191.567 9439850.643 GAR 307282.3055 148353.8573 348774.8869 556098.368 351447.1368 532178.3901 950590.2352 660656.2144 511471.9582 315108.9818 58086.02966 45633.42018 292189.0507 499545.5784 139208.191 229912.251 GDP 3601060.485 2293506.831 5053424.548 3271673.37 3416080.673 1864023.967 6335700.026 2363615.878 3682240.151 6602775.811 184681.6081 1303577.315 1985166.62 3119226.82 3466743.343 2258869.513 glucose 2948103.422 3144579.219 4241012.438 3508300.313 4413671.25 3911602.656 1.54E+07 1.29E+07 1.05E+07 1.29E+07 1535648.336 2444257.328 3362657.781 6651844.25 3446354.219 4422565.438 glucose-6-phosphate 6006072.676 1331014.935 9747527.458 6042379.552 9268819.796 3357934.97 1.51E+07 1.02E+07 3853685.892 3633156.264 1125895.656 687605.6658 1.13E+07 5559396.376 3150615.497 1631657.605 glutamate 4.25E+08 3.13E+08 5.39E+08 3.52E+08 4.99E+08 4.07E+08 6.90E+08 4.18E+08 4.88E+08 5.16E+08 1.36E+08 2.39E+08 4.22E+08 5.01E+08 3.76E+08 3.63E+08 glutamine 1.74E+08 9.65E+07 1.46E+08 9.41E+07 2.36E+08 1.35E+08 2.90E+08 1.45E+08 2.91E+08 3.27E+08 7.00E+07 9.09E+07 1.68E+08 1.99E+08 2.51E+08 1.30E+08 glyceraldehyde-3-phosphate 1049519.671 2230052.81 3327187.48 1352166.491 2588538.671 2564251.434 4550369.36 1588453.922 3353854.247 9705092.187 351104.5064 956631.7233 882498.5742 1695499.987 816291.4987 1964562.246 glycerol-3-phosphate 1.46E+07 1.16E+07 1.87E+07 1.30E+07 1.60E+07 1.40E+07 3.10E+07 1.70E+07 1.72E+07 1.62E+07 3279946.09 3757233.448 1.04E+07 2.02E+07 1.68E+07 2.02E+07 GSH 1.91E+08 7.44E+07 2.48E+08 1.51E+08 2.97E+08 1.56E+08 3.23E+08 1.51E+08 1.65E+08 2.43E+08 1712854.514 1.54E+07 1.39E+08 2.04E+08 1.63E+08 1.62E+08 GSSG 4190869.336 4631586.965 4825326.583 3342727.343 6206149.35 4310488.422 2966549.058 3062823.406 4767883.946 4550530.117 1181654.334 5489351.037 4703653.617 5643155.672 5308427.401 5498131.681 GTP 635000.3323 269910.2402 920925.7367 584287.8185 598613.011 323023.8096 913303.9489 353355.0912 734038.6028 1907969.975 41439.37503 439813.6201 304831.4856 479916.9871 907902.9786 319775.1229 guanosine 571488.6057 529053.4683 1001899.327 661577.503 2477424.275 1426704.018 1030546.615 761634.2441 485462.3974 461927.6804 647015.7392 185259.2432 400881.5234 464487.6446 205174.3353 588674.3185 histidine 2.67E+07 2.36E+07 3.62E+07 3.06E+07 5.21E+07 4.23E+07 4.52E+07 2.90E+07 4.87E+07 6.27E+07 1.42E+07 2.15E+07 2.94E+07 4.76E+07 3.17E+07 4.29E+07 hydroxyglutarate 2.60E+07 3.83E+07 3.08E+07 2.22E+07 1.90E+07 2.05E+07 2.75E+07 2.42E+07 2.94E+07 2.75E+07 1.42E+07 3.53E+07 3.29E+07 4.32E+07 5.12E+07 5.50E+07 hypotaurine 4783193.994 3966771.22 8503960.77 4940537.524 4889540.163 3774399.788 8385759.005 3595781.802 3593302.301 5286426.197 1465444.456 3398269.833 3022337.53 5691812.729 2835663.568 2461653.3 IMP 1804083.301 1577577.438 2953682.594 1891643.427 2479924.901 3028513.999 2703551.566 4128440.288 2189338.969 1698517.64 591043.4031 749331.0877 2314254.593 3928488.436 2452652.138 2594134.164 inosine 5407879.71 4643739.002 9160087.365 6675318.504 2.39E+07 1.49E+07 1.12E+07 1.13E+07 6122012.389 3657405.864 6581279.304 1013333.163 1.06E+07 6859974.61 3234251.195 6913159.634 lactate 5.70E+08 3.82E+08 9.61E+08 6.14E+08 1.10E+09 6.65E+08 1.37E+09 6.32E+08 6.53E+08 7.93E+08 2.58E+08 1.48E+08 6.54E+08 8.91E+08 7.98E+08 8.93E+08 lauric acid 1.13E+08 1.40E+08 9.23E+07 1.15E+08 1.30E+08 6.92E+07 6.93E+07 9.05E+07 1.19E+08 7.05E+07 1.45E+08 7.40E+07 6.89E+07 3.28E+08 1.01E+08 6.84E+07 malate 2.91E+08 1.84E+08 4.42E+08 2.64E+08 3.76E+08 2.79E+08 5.23E+08 2.98E+08 3.50E+08 3.03E+08 1.15E+08 1.03E+08 3.25E+08 3.42E+08 3.38E+08 3.12E+08 myristic acid 7.38E+08 4.17E+08 5.38E+08 8.18E+08 6.53E+08 4.87E+08 5.48E+08 5.36E+08 6.13E+08 5.19E+08 6.08E+08 5.76E+08 1.01E+09 6.07E+08 7.81E+08 9.80E+08 N-acetylglucosamine-phosphate 735650.0703 764736.6426 1122270.209 966879.4669 1420308.047 1310878.047 1627480.885 1450605.391 1138981.275 861073.1067 1011972.582 342372.3359 633063.2967 1019406.819 716890.9311 1479649.967 N-acetylglutamate 1.69E+07 4783698.681 7233459.972 4709434.182 2.02E+07 1.38E+07 9786899.923 6956666.083 9672638.378 1.55E+07 351156.4868 1309685.507 3138531.087 7599103.576 3032763.201 2956266.874 NADH 2288699.846 1028268.229 1786524.197 1338326.674 1942684.953 1284721.202 2634609.375 1159796.156 1626542.248 1364162.275 210446.7152 400489.3136 2364836.154 2052848.597 2640519.122 1135069.834 oleic acid 9.78E+07 8.20E+07 4.84E+07 2.90E+08 5.52E+07 9.43E+07 5.33E+07 1.18E+08 9.18E+07 4.20E+07 2.37E+08 1.14E+08 5.86E+07 5.46E+07 4.27E+07 1.34E+08 orotate 960385.3969 3205093.283 1814363.22 1408076.241 708066.0326 1277412.054 1505348.822 1880082.694 1025687.026 1210591.505 2366407 1823295.343 2045926.418 3719377.558 2689519.316 7058785.133 palmitic acid 5.42E+08 5.05E+08 3.70E+08 5.97E+08 4.42E+08 4.28E+08 3.48E+08 4.20E+08 4.54E+08 3.54E+08 6.74E+08 5.36E+08 4.53E+08 3.69E+08 4.86E+08 5.87E+08 phosphate 6.75E+07 6.33E+07 6.89E+07 7.33E+07 6.58E+07 6.54E+07 5.50E+07 6.61E+07 6.86E+07 4.56E+07 7.95E+07 6.39E+07 6.49E+07 6.12E+07 5.70E+07 6.54E+07 phosphatidylethanolamine 0 0 0 0 0 0 0 30559.34802 31467.58774 0 0 0 0 0 0 54645.50395 phosphocreatine 5069383.819 1801678.17 8258689.591 4244030.274 2915114.588 1135747.779 4861520.035 1298617.649 1949415.994 3424038.95 1809761.703 5962461.568 2657512.533 4729990.956 2840223.084 1953033.287 phosphoenolpyruvate 965970.2009 1581174.631 1371849.164 2890336.69 1233226.583 3030605.349 1121425.185 2729393.994 1334663.307 4412523.531 357595.7707 382178.3925 221197.8253 1625156.008 1014031.354 1845460.199 phosphoglycerate 3221102.724 1732521.651 2676704.411 2852945.965 3095133.01 2444419.373 2459666.278 2430674.276 3181702.307 2659681.136 2076039.299 1993096.745 2634630.685 1997684.833 3240027.344 1789071.023 phosphoribosylamine 58199.08162 36175.74354 55441.78527 49494.02841 72521.83598 78022.0186 61835.60961 93859.63258 57440.43621 0 28142.79924 21270.89533 79767.58255 72417.5569 53521.41659 60265.53012 phosphorylethanolamine 1.23E+08 8.97E+07 1.86E+08 1.11E+08 1.52E+08 1.15E+08 2.34E+08 1.15E+08 1.13E+08 1.72E+08 2.88E+07 5.02E+07 3.29E+07 9.04E+07 5.13E+07 5.78E+07 phosphoserine 65506.41252 38123.57986 90348.63938 37985.01189 55619.71058 35320.78278 73830.95958 40545.63601 56736.07739 82969.27905 0 86266.80004 68099.84972 83225.93905 63409.59566 55235.60341 pyruvate 951229.2979 633697.4982 1712423.091 956477.8023 1485662.443 1313179.316 2778189.916 1202461.348 2028646.689 4199048.739 900008.0093 824570.5728 1029994.849 1808964.631 1183425.877 1655581.133 quinolinic acid 1443830.517 1536109.833 1783907.685 1340531.791 977953.4198 1178808.685 1771566.062 1223173.724 818913.497 1381479.826 823193.9176 1076382.821 1393057.378 3264251.252 1156036.661 1319669.403 rib/ribul/xylul-ose-phosphate 2156992.04 1656302.491 2189434.273 2125178.78 2881728.158 3550296.406 2593931.68 4674373.154 2792199.453 2103423.449 1925868.052 863687.759 3471919.67 2494016.698 2420295.588 2647827.788 S-(1;2-Dicarboxyethyl)glutathione 99810.06979 50539.24687 165441.5899 102524.3145 146300.7682 70864.60686 160382.4703 94532.08545 88800.6525 99621.63173 0 28252.23618 116816.7864 121877.1921 92698.21239 104612.8535 S-(2-Succinyl)-L-cysteine 356698.1827 370989.6054 601370.7378 541752.2523 744036.6978 558261.595 686602.2311 579333.9372 407082.8451 1153375.777 124122.9953 285905.4358 520670.4982 942048.2637 391659.0734 660628.8611 saccharopine 3207192.558 2962856.959 4408834.165 3194885.672 8369482.741 4568982.684 4931082.412 2493739.397 4152331.926 4026472.406 375780.8934 687827.3039 807862.1626 1604121.78 2028515.807 3159562.089 S-adenosyl-homocysteine 546455.7225 391089.4694 658431.5521 539068.7905 657049.8663 407463.3295 616430.874 424205.5156 372988.6427 492816.7897 226710.039 197329.3318 514560.2256 551144.2217 421321.5244 493007.8589 sedoheptulose-7-phosphate 2430563.477 1039595.308 5137029.736 3101239.749 3751876.124 2591406.766 1.03E+07 3947137.262 2655937.571 2829584.429 707779.7924 380026.6973 4799913.911 3179563.646 1293820.179 1526376.537 sorbitol 791008.9098 432817.1485 1544213.192 938129.4295 1934832.793 114067.1157 2948654.074 948260.8407 1048571.74 1391469.426 23785.85498 114284.5092 676657.0604 865416.06 71773.59126 486693.7188 stearic acid 3.13E+08 3.32E+08 2.43E+08 2.95E+08 2.60E+08 3.35E+08 2.08E+08 2.80E+08 3.08E+08 1.85E+08 6.86E+08 4.87E+08 2.78E+08 2.44E+08 2.26E+08 3.23E+08 succinate 4.72E+07 3.93E+07 7.19E+07 4.12E+07 6.37E+07 3.02E+07 8.53E+07 3.31E+07 2.81E+07 2.60E+07 3.08E+07 2.80E+07 5.18E+07 7.23E+07 6.35E+07 8.26E+07 taurine 4.31E+08 3.00E+08 5.13E+08 3.37E+08 2.96E+08 3.34E+08 4.93E+08 3.87E+08 2.93E+08 3.27E+08 1.30E+08 2.29E+08 3.72E+08 6.58E+08 3.12E+08 2.97E+08 trifluoromethanesulfonate 3.70E+07 5.01E+07 4.19E+07 4.38E+07 4.36E+07 4.14E+07 4.30E+07 5.09E+07 4.92E+07 4.66E+07 4.58E+07 5.53E+07 4.61E+07 4.80E+07 4.51E+07 4.17E+07 tryptophan 4546639.929 4211720.552 7631710.451 5120138.922 9877122.86 8425230.938 7960886.212 5012759.294 4338139.869 4207839.392 5062572.003 1688998.529 3844335.06 5640067.382 4619855.393 8285939.289 tyrosine 7061630.267 5190061.398 1.11E+07 7627450.186 1.39E+07 1.10E+07 1.64E+07 1.01E+07 7408426.175 7907618.051 5352899.967 2220192.433 5700121.448 8063897.657 7108261.556 1.22E+07 UDP-glucuronic acid 3851378.336 3076075.318 7988672.905 4428555.114 1.04E+07 5110838.527 8128893.065 4104784.043 4506522.454 6239400.31 807576.0498 1685658.425 4718056.252 6768983.378 4602868.594 3472248.341 UDP-N-acetylglucosamine 3.81E+07 2.83E+07 7.31E+07 3.83E+07 1.00E+08 5.21E+07 9.33E+07 4.48E+07 5.34E+07 1.06E+08 6538943.236 1.28E+07 2.75E+07 4.52E+07 3.31E+07 3.40E+07 UMP 4.15E+07 2.49E+07 5.60E+07 3.40E+07 5.88E+07 3.58E+07 5.81E+07 3.50E+07 3.21E+07 4.66E+07 8473549.013 1.45E+07 4.82E+07 6.56E+07 2.63E+07 3.98E+07 uric acid 2.35E+07 2.63E+07 2.54E+07 1.94E+07 2.70E+07 2.99E+07 3.27E+07 3.90E+07 2.70E+07 2.99E+07 1.51E+07 1.60E+07 1.50E+07 2.63E+07 2.03E+07 3.55E+07 UTP 2102449.184 342038.7148 3304865.896 1716984.61 2143603.568 768893.9294 2832957.184 838311.153 1740609.33 4722039.21 130950.9951 1106625.98 1696015.228 2090909.066 2756709.721 1109160.524 XMP 40082.79467 75984.2781 0 35173.76985 17552.09752 39493.18908 22013.75338 65262.4199 47464.07386 11255.20357 39803.34916 47707.71624 34221.60851 66827.00802 43727.36674 73775.17942 xylonic acid 2251496.995 3584823.669 5374362.004 4132938.448 3208173.511 3814771.048 7014158.423 4536154.849 4543772.714 6654400.932 1462463.288 2724194.134 4032492.275 7128473.719 3625625.629 4428423.58 xylulose 299358.6027 288961.6009 444756.0729 288600.4934 521091.6323 391877.712 271444.9719 206285.3559 140431.4429 199576.9998 111759.9728 116068.8523 214903.4081 316778.9207 295197.1072 344046.8339 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z retention time, min PubChem ID 6-phosphogluconic acid 275.017 10.82 91493 acetyl-CoA 808.119 6.75 444493 adenylosuccinic acid 462.067 11.29 447145 ADP 426.022 8.77 6022 AICAR 257.089 12.85 90471726 alpha-ketoglutarate 145.014 8.97 51 AMP 346.056 6.98 6083 arachidic acid 311.296 1.52 10467 argininosuccinate 289.115 9.7 439998 ascorbate 175.025 7.62 54670067 aspartate 132.03 8.3 5960 ATP 505.988 9.8 5957 CAIR 338.039 3.93 165388 carbamoyl-aspartate 175.036 9.91 279 CDP 402.011 10.18 6132 CDP-ethanolamine 445.053 9.43 123727 cis-aconitate 173.009 10.87 643757 citrate / isocitrate 191.02 11.02 311 CMP 322.045 8.71 6131 CoA 766.108 7.76 87642 CTP 481.977 11.22 6176 cysteine 120.012 8.82 5862 cystine 239.017 8.82 595 cytidine 242.078 5.49 6175 dCTP 465.982 10.36 65091 dihydroorotate 157.025 5.7 5461056 dihydroxyacetone-phosphate 168.991 8.82 668 dTMP 321.049 6.98 9700 dTTP 480.982 9.36 64968 FGAR 313.044 8.74 130805 fructose 179.056 6.74 2723872 fructose-1;6-bisphosphate 338.989 11.18 10267 fructose-1-phosphate 259.022 8.68 65246 fructose-6-phosphate 259.022 9.37 69507 fumarate 115.004 9.37 444972 gamma-amino-butyrate 102.056 8.1 119 GAR 285.049 6.91 160913 GDP 442.017 10.87 135398619 glucose 179.056 7.35 5793 glucose-6-phosphate 259.022 9.96 5958 glutamate 146.046 8.1 33032 glutamine 145.062 7.65 5961 glyceraldehyde-3-phosphate 168.991 9.42 729 glycerol-3-phosphate 171.006 8.56 754 GSH 306.077 7.51 124886 GSSG 611.145 10.55 65359 GTP 521.983 11.82 135398633 guanosine 282.084 6.08 135398635 histidine 154.062 7.35 6274 hydroxyglutarate 147.03 8.77 43 hypotaurine 108.012 7.6 107812 IMP 347.04 8.84 135398640 inosine 267.073 4.99 135398641 lactate 89.024 4.41 612 lauric acid 199.17 1.65 3893 malate 133.014 9.37 525 myristic acid 227.202 1.61 11005 N-acetylglucosamine-phosphate 300.049 8.45 439219 N-acetylglutamate 188.056 8.06 70914 NADH 664.118 7.44 439153 oleic acid 281.249 1.56 445639 orotate 155.01 4.99 1492348 palmitic acid 255.233 1.56 985 phosphate 96.97 7.11 1004 phosphatidylethanolamine 298.07 8.82 5327011 phosphocreatine 210.029 8.77 9548602 phosphoenolpyruvate 166.975 10.55 1005 phosphoglycerate 184.986 10.12 439183 phosphoribosylamine 228.028 9.02 150 phosphorylethanolamine 140.012 8.97 1015 phosphoserine 184.002 9.7 68841 pyruvate 87.009 3.58 1060 quinolinic acid 166.015 3.89 1066 rib/ribul/xylul-ose-phosphate 229.012 9.07 439190 S-(1;2-Dicarboxyethyl)glutathione 422.087 11.45 329770102 S-(2-Succinyl)-L-cysteine 236.023 10.39 N/A saccharopine 275.125 8.87 160556 S-adenosyl-homocysteine 383.114 6.59 439155 sedoheptulose-7-phosphate 289.033 9.48 22833559 sorbitol 181.072 6.86 5780 stearic acid 283.264 1.56 5281 succinate 117.019 8.82 1110 taurine 124.007 7.55 1123 trifluoromethanesulfonate 148.953 1.92 2758875 tryptophan 203.083 5.24 6305 tyrosine 180.067 6.24 6057 UDP-glucuronic acid 579.027 11.78 17473 UDP-N-acetylglucosamine 606.074 8.77 445675 UMP 323.029 8.69 6030 uric acid 167.021 6.81 1175 UTP 482.961 10.87 6133 XMP 363.035 10.97 10287649 xylonic acid 165.04 6.81 6602431 xylulose 149.046 5.11 5289590 METABOLITES_END #END