#METABOLOMICS WORKBENCH jer608_20230906_172834 DATATRACK_ID:4289 STUDY_ID:ST002847 ANALYSIS_ID:AN004665 PROJECT_ID:PR001782 VERSION 1 CREATED_ON 08-21-2024 #PROJECT PR:PROJECT_TITLE Targeting Pancreatic Cancer Metabolic Dependencies through Glutamine Antagonism. PR:PROJECT_TYPE Manuscript PR:PROJECT_SUMMARY Pancreatic ductal adenocarcinoma (PDAC) cells utilize glutamine (Gln) to support PR:PROJECT_SUMMARY proliferation and redox balance. Earlier attempts to inhibit Gln metabolism PR:PROJECT_SUMMARY using glutaminase inhibitors resulted in rapid metabolic reprogramming and PR:PROJECT_SUMMARY therapeutic resistance. We demonstrated that treating PDAC cells with a Gln PR:PROJECT_SUMMARY antagonist, 6-Diazo-5-oxo-L-norleucine (DON), led to a metabolic crisis in PR:PROJECT_SUMMARY vitro. In addition, we observed a profound decrease in tumor growth in various PR:PROJECT_SUMMARY in vivo models using DRP-104 (sirpiglenastat), a pro-drug version of DON PR:PROJECT_SUMMARY designed to circumvent DON-associated toxicity. PR:INSTITUTE New York University PR:DEPARTMENT Radiation Oncology PR:LABORATORY Alec C Kimmelman PR:LAST_NAME Encarnacion Rosado PR:FIRST_NAME Joel PR:ADDRESS Smilow Research Building Room 907G New York, NY 10016 PR:EMAIL jencarnacionrosado@salk.edu, Alec.Kimmelman@nyulangone.org PR:PHONE 646-501-8984 PR:DOI http://dx.doi.org/10.21228/M8CB14 #STUDY ST:STUDY_TITLE Targeting Pancreatic Cancer Metabolic Dependencies through Glutamine Antagonism. ST:STUDY_SUMMARY Pancreatic ductal adenocarcinoma (PDAC) cells utilize glutamine (Gln) to support ST:STUDY_SUMMARY proliferation and redox balance. Earlier attempts to inhibit Gln metabolism ST:STUDY_SUMMARY using glutaminase inhibitors resulted in rapid metabolic reprogramming and ST:STUDY_SUMMARY therapeutic resistance. Here, we demonstrated that treating PDAC cells with a ST:STUDY_SUMMARY Gln antagonist, 6-Diazo-5-oxo-L-norleucine (DON), led to a metabolic crisis in ST:STUDY_SUMMARY vitro. In addition, we observed a profound decrease in tumor growth in various ST:STUDY_SUMMARY in vivo models using DRP-104 (sirpiglenastat), a pro-drug version of DON that ST:STUDY_SUMMARY was designed to circumvent DON associated toxicity. We found that ERK signaling ST:STUDY_SUMMARY is increased as a compensatory mechanism. Combinatorial treatment of DRP-104 and ST:STUDY_SUMMARY Trametinib led to a significant increase in survival in a syngeneic model PDAC. ST:STUDY_SUMMARY These proof-of-concept studies suggested that broadly targeting Gln metabolism ST:STUDY_SUMMARY could provide a therapeutic avenue for PDAC. The combination with an ERK ST:STUDY_SUMMARY signaling pathway inhibitor could further improve the therapeutic outcome. ST:INSTITUTE New York University ST:LAST_NAME Encarnacion Rosado ST:FIRST_NAME Joel ST:ADDRESS Smilow Research Building Room 907G New York, NY 10016 ST:EMAIL jencarnacionrosado@salk.edu, Alec.Kimmelman@nyulangone.org ST:PHONE 646-501-8984 ST:SUBMIT_DATE 2023-09-06 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Not applicable #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - HY19_DON_1 Vehicle:DON RAW_FILE_NAME=S23449.mzML SUBJECT_SAMPLE_FACTORS - HY19_DON_2 Vehicle:DON RAW_FILE_NAME=S23450.mzML SUBJECT_SAMPLE_FACTORS - HY19_DON_3 Vehicle:DON RAW_FILE_NAME=S23451.mzML SUBJECT_SAMPLE_FACTORS - HY19_DRP104_1 Vehicle:DRP-104 RAW_FILE_NAME=S23452.mzML SUBJECT_SAMPLE_FACTORS - HY19_DRP104_2 Vehicle:DRP-104 RAW_FILE_NAME=S23453.mzML SUBJECT_SAMPLE_FACTORS - HY19_DRP104_3 Vehicle:DRP-104 RAW_FILE_NAME=S23454.mzML SUBJECT_SAMPLE_FACTORS - HY19_CNT_1 Vehicle:Vehicle RAW_FILE_NAME=S23446.mzML SUBJECT_SAMPLE_FACTORS - HY19_CNT_2 Vehicle:Vehicle RAW_FILE_NAME=S23447.mzML SUBJECT_SAMPLE_FACTORS - HY19_CNT_3 Vehicle:Vehicle RAW_FILE_NAME=S23448.mzML #COLLECTION CO:COLLECTION_SUMMARY As described in Parker, et al (https://doi.org/10.1038/s41467-021-25228-9), CO:COLLECTION_SUMMARY xtraction of metabolites from cell pellets— Metabolites were initially CO:COLLECTION_SUMMARY extracted from samples by quickly aspirating the cell culture media and adding CO:COLLECTION_SUMMARY 1 mL of extraction buffer, consisting of 80% methanol (Fisher Scientific) and CO:COLLECTION_SUMMARY 500 nM metabolomics amino acid mix standard (Cambridge Isotope Laboratories). CO:COLLECTION_SUMMARY To effectively scale all harvested samples to equivalent volumes of extraction CO:COLLECTION_SUMMARY buffer, samples were fully dried down by Speedvac (Thermo Fisher, Waltham, MA) CO:COLLECTION_SUMMARY and reconstituted volumetrically by mixing the entire dried cell pellet sample CO:COLLECTION_SUMMARY with 1 mL of 80% methanol without QC standards in 2.0 mL screw cap vials CO:COLLECTION_SUMMARY containing ~100 µL of disruption beads (Research Products International, CO:COLLECTION_SUMMARY Mount Prospect, IL). Samples were scaled to a ratio of 1e6 cells to 1 mL of CO:COLLECTION_SUMMARY extraction solvent with all steps being carried out in a cold room. Each was CO:COLLECTION_SUMMARY homogenized for 10 cycles on a bead blaster homogenizer (Benchmark Scientific, CO:COLLECTION_SUMMARY Edison, NJ). Cycling consisted of a 30 sec homogenization time at 6 m/s CO:COLLECTION_SUMMARY followed by a 30 sec pause. Samples were subsequently spun at 21,000 × g CO:COLLECTION_SUMMARY for 3 min at 4 °C. A set volume of each (450 µL) was transferred to a CO:COLLECTION_SUMMARY 1.5 mL tube and dried down by Speedvac concentration. Samples were CO:COLLECTION_SUMMARY reconstituted in 50 µL of Optima LC/MS grade water (Fisher Scientific, CO:COLLECTION_SUMMARY Waltham, MA). Samples were sonicated for 2 min, then centrifuged at CO:COLLECTION_SUMMARY 21,000 × g for 3 min at 4 °C. Twenty microliters were transferred to CO:COLLECTION_SUMMARY LC vials containing glass inserts for analysis. The remaining sample was placed CO:COLLECTION_SUMMARY at −80 °C for long-term storage. Samples were subjected to an LC-MS CO:COLLECTION_SUMMARY analysis to detect and quantify known peaks. A metabolite extraction was carried CO:COLLECTION_SUMMARY out on each sample by quickly aspirating experimental media and adding 1 mL of CO:COLLECTION_SUMMARY 80% methanol containing internal QC standards. A MilliporeTM ZIC-pHILIC CO:COLLECTION_SUMMARY (2.1 × 150 mm, 5 μm) LC column was coupled to a Dionex Ultimate 3000TM CO:COLLECTION_SUMMARY system. The column oven temperature and flow rate were set to 25 °C and CO:COLLECTION_SUMMARY 100 μL/min, respectively, for the following gradient elution: 80–20%B CO:COLLECTION_SUMMARY (0–30 min), 20–80%B (30–31 min), 80–80%B (31–42 min). Mobile CO:COLLECTION_SUMMARY phase compositions were: (A) 10 mM ammonium carbonate in water, pH 9.0 and (B) CO:COLLECTION_SUMMARY neat acetonitrile; and an injection volume of 2 μL was used for all analyses. CO:COLLECTION_SUMMARY The LC system was coupled to a Thermo Q Exactive HFTM mass spectrometer CO:COLLECTION_SUMMARY operating in heated electrospray ionization mode (HESI) for LC-MS analysis. A CO:COLLECTION_SUMMARY 30-m polarity switching data-dependent Top 5 method was used for both positive CO:COLLECTION_SUMMARY and negative modes. The following parameters were also set: spray voltage of CO:COLLECTION_SUMMARY 3.5 kV, capillary temperature at 320 °C, sheath gas flow rate of 35, aux CO:COLLECTION_SUMMARY gas rate of 10, and max spray current of 100 μA. Full MS scan parameters for CO:COLLECTION_SUMMARY both positive and negative modes were set as followed: scan range of CO:COLLECTION_SUMMARY 67–1000 m/z, resolution of 120,000, AGC target of 3e6, and maximum IT of CO:COLLECTION_SUMMARY 100 ms. Tandem MS spectra for both positive and negative modes used a CO:COLLECTION_SUMMARY resolution of 15,000, fixed first mass of 50 m/z, isolation window of CO:COLLECTION_SUMMARY 0.4 m/z, isolation offset of 0.1 m/z, AGC target of 1e5, minimum AGC target CO:COLLECTION_SUMMARY of 1e4, intensity threshold of 2e5, maximum IT of 50 ms, and three-way CO:COLLECTION_SUMMARY multiplexed normalized collision energies (nCE) of 10, 30, 80. All data were CO:COLLECTION_SUMMARY acquired in profile mode. ThermoTM RAW files were read using ThermoFisher CO:COLLECTION_SUMMARY CommonCore RawFileReader. An in-house python script (Skeleton) was used for CO:COLLECTION_SUMMARY detection and quantification of sample peaks and internal standards based on a CO:COLLECTION_SUMMARY retention time and accurate mass library adapted from the Whitehead Institute94 CO:COLLECTION_SUMMARY and verified with authentic standards and/or high-resolution MS/MS spectra CO:COLLECTION_SUMMARY manually curated using the NIST14MS/MS95 and METLIN (2017)96 tandem mass CO:COLLECTION_SUMMARY spectral libraries. For feature-based analysis, an in-house python script CO:COLLECTION_SUMMARY (Ungrid) was used to detect MS1 peaks across all samples using the following CO:COLLECTION_SUMMARY parameters: a m/z discrimination threshold of 20 ppm, a minimum peak intensity CO:COLLECTION_SUMMARY of 1e5, a minimum signal-to-noise ratio of 10, and a retention time threshold of CO:COLLECTION_SUMMARY 2 min. Metabolite and feature peaks extracted in this manner were defined by CO:COLLECTION_SUMMARY either the detected feature m/z or the theoretical m/z of the expected ion type CO:COLLECTION_SUMMARY for the standard in the library (e.g., [M+H]+). The following parameters were CO:COLLECTION_SUMMARY applied: a ±5 part-per-million (ppm) tolerance, an initial retention time CO:COLLECTION_SUMMARY search window of ±0.5 min across all samples, and a ±7.5 s peak apex CO:COLLECTION_SUMMARY retention time tolerance within individual samples. An in-house statistical CO:COLLECTION_SUMMARY pipeline, Metabolize (version 1.0), was used to process the resulting data CO:COLLECTION_SUMMARY matrix of metabolite intensities for all samples and blank controls. A final CO:COLLECTION_SUMMARY peak detection was calculated based on a signal-to-noise ratio (S/N) or 3× CO:COLLECTION_SUMMARY blank controls with a floor of 1e5 (arb. units). The threshold value was input CO:COLLECTION_SUMMARY for any sample where the calculated peak intensity was lower than the blank CO:COLLECTION_SUMMARY threshold for any statistical comparisons. The resulting blank corrected data CO:COLLECTION_SUMMARY matrix was used for all group-wise comparisons. T-tests were performed using the CO:COLLECTION_SUMMARY Python SciPy library (version 1.1.0)97 to test for differences and generate CO:COLLECTION_SUMMARY statistics. Any metabolite with p-value < 0.05 was considered CO:COLLECTION_SUMMARY significantly regulated (up or down). Volcano plots were generated utilizing CO:COLLECTION_SUMMARY Prism (GraphPad). The R package DESeq2 (1.24.0)98 was used to adjust for CO:COLLECTION_SUMMARY covariate effects (as applicable) and to calculate the adjusted p-value in the CO:COLLECTION_SUMMARY covariate model. Zero values were input for non-detected values instead of the CO:COLLECTION_SUMMARY blank threshold to avoid false positive. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY HY19636 cells were plated in a six-well plate at 2.0x10^5 cells/well and allowed TR:TREATMENT_SUMMARY to attach overnight in DMEM. Next, cells were washed with PBS twice and cultured TR:TREATMENT_SUMMARY for 24 hours in DMEM supplemented with 10% dialyzed serum was added. Cells were TR:TREATMENT_SUMMARY pre-treated with DON (25µM) or DRP-104 (25µM) overnight, media was removed and TR:TREATMENT_SUMMARY washed with PBS. Then, cells were frozen in -80C and until metabolite extraction #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Extraction of metabolites from cell pellets— Metabolites were initially SP:SAMPLEPREP_SUMMARY extracted from samples by quickly aspirating the cell culture media and adding SP:SAMPLEPREP_SUMMARY 1 mL of extraction buffer, consisting of 80% methanol (Fisher Scientific) and SP:SAMPLEPREP_SUMMARY 500 nM metabolomics amino acid mix standard (Cambridge Isotope Laboratories). SP:SAMPLEPREP_SUMMARY To effectively scale all harvested samples to equivalent volumes of extraction SP:SAMPLEPREP_SUMMARY buffer, samples were fully dried down by Speedvac (Thermo Fisher, Waltham, MA) SP:SAMPLEPREP_SUMMARY and reconstituted volumetrically by mixing the entire dried cell pellet sample SP:SAMPLEPREP_SUMMARY with 1 mL of 80% methanol without QC standards in 2.0 mL screw cap vials SP:SAMPLEPREP_SUMMARY containing ~100 µL of disruption beads (Research Products International, SP:SAMPLEPREP_SUMMARY Mount Prospect, IL). Samples were scaled to a ratio of 1e6 cells to 1 mL of SP:SAMPLEPREP_SUMMARY extraction solvent with all steps being carried out in a cold room. Each was SP:SAMPLEPREP_SUMMARY homogenized for 10 cycles on a bead blaster homogenizer (Benchmark Scientific, SP:SAMPLEPREP_SUMMARY Edison, NJ). Cycling consisted of a 30 sec homogenization time at 6 m/s SP:SAMPLEPREP_SUMMARY followed by a 30 sec pause. Samples were subsequently spun at 21,000 × g SP:SAMPLEPREP_SUMMARY for 3 min at 4 °C. A set volume of each (450 µL) was transferred to a SP:SAMPLEPREP_SUMMARY 1.5 mL tube and dried down by Speedvac concentration. Samples were SP:SAMPLEPREP_SUMMARY reconstituted in 50 µL of Optima LC/MS grade water (Fisher Scientific, SP:SAMPLEPREP_SUMMARY Waltham, MA). Samples were sonicated for 2 min, then centrifuged at SP:SAMPLEPREP_SUMMARY 21,000 × g for 3 min at 4 °C. Twenty microliters were transferred to SP:SAMPLEPREP_SUMMARY LC vials containing glass inserts for analysis. The remaining sample was placed SP:SAMPLEPREP_SUMMARY at −80 °C for long-term storage. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) CH:COLUMN_TEMPERATURE 25 CH:FLOW_GRADIENT The gradient profile was as follows; 80-20%B (0-30 min), 20-80%B (30-31 min), CH:FLOW_GRADIENT 80-80%B (31-42 min) CH:FLOW_RATE 100 uL/min CH:SOLVENT_A 10 mM ammonium carbonate in water, pH 9.0 CH:SOLVENT_B acetonitrile CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS The LC system was coupled to a Thermo Q Exactive HFTM mass spectrometer MS:MS_COMMENTS operating in heated electrospray ionization mode (HESI) for LC-MS analysis. A MS:MS_COMMENTS 30-m polarity switching data-dependent Top 5 method was used for both positive MS:MS_COMMENTS and negative modes. The following parameters were also set: spray voltage of MS:MS_COMMENTS 3.5 kV, capillary temperature at 320 °C, sheath gas flow rate of 35, aux MS:MS_COMMENTS gas rate of 10, and max spray current of 100 μA. Full MS scan parameters for MS:MS_COMMENTS both positive and negative modes were set as followed: scan range of MS:MS_COMMENTS 67–1000 m/z, resolution of 120,000, AGC target of 3e6, and maximum IT of MS:MS_COMMENTS 100 ms. Tandem MS spectra for both positive and negative modes used a MS:MS_COMMENTS resolution of 15,000, fixed first mass of 50 m/z, isolation window of MS:MS_COMMENTS 0.4 m/z, isolation offset of 0.1 m/z, AGC target of 1e5, minimum AGC target MS:MS_COMMENTS of 1e4, intensity threshold of 2e5, maximum IT of 50 ms, and three-way MS:MS_COMMENTS multiplexed normalized collision energies (nCE) of 10, 30, 80. All data were MS:MS_COMMENTS acquired in profile mode. ThermoTM RAW files were read using ThermoFisher MS:MS_COMMENTS CommonCore RawFileReader. An in-house python script (Skeleton) was used for MS:MS_COMMENTS detection and quantification of sample peaks and internal standards based on a MS:MS_COMMENTS retention time and accurate mass library adapted from the Whitehead Institute94 MS:MS_COMMENTS and verified with authentic standards and/or high-resolution MS/MS spectra MS:MS_COMMENTS manually curated using the NIST14MS/MS95 and METLIN (2017)96 tandem mass MS:MS_COMMENTS spectral libraries. MS:ION_MODE UNSPECIFIED #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion counts MS_METABOLITE_DATA_START Samples HY19_DON_1 HY19_DON_2 HY19_DON_3 HY19_DRP104_1 HY19_DRP104_2 HY19_DRP104_3 HY19_CNT_1 HY19_CNT_2 HY19_CNT_3 Factors Vehicle:DON Vehicle:DON Vehicle:DON Vehicle:DRP-104 Vehicle:DRP-104 Vehicle:DRP-104 Vehicle:Vehicle Vehicle:Vehicle Vehicle:Vehicle 1-Methylnicotinamide 82956.0000 59250.0000 52213.0000 63795.0000 2_3-Diphosphoglyceric acid 3-Hydroxybutyric acid 3-Phosphoglyceric acid 2360461.0000 3853256.0000 2724835.0000 1706643.0000 3380644.0000 2005825.0000 6498588.0000 5386352.0000 3090533.0000 4_5-Dihydroorotic acid 4-Hydroxyproline 107326.0000 133595.0000 204345.0000 151346.0000 187137.0000 229745.0000 130776.0000 150598.0000 5^-Methylthioadenosine 146934.0000 151846.0000 144796.0000 138294.0000 124009.0000 94689.0000 96149.0000 5-Phosphoribosylamine 17891.0000 15094.0000 16775.0000 12341.0000 11340.0000 17522.0000 21991.0000 25390.0000 23580.0000 6-Phosphogluconic acid 22139.0000 19220.0000 27929.0000 Acetoacetic acid 41463.0000 Acetyl-CoA Acetylcysteine 34667.0000 21384.0000 17945.0000 28871.0000 15669.0000 27542.0000 17623.0000 14376.0000 Adenine 16496442.0000 15120138.0000 14856874.0000 489019.0000 614821.0000 18406650.0000 28721482.0000 26043286.0000 Adenosine 28081984.0000 4786747.0000 8067276.0000 4901472.0000 2537381.0000 5493814.0000 1511104.0000 5815696.0000 Adenosine diphosphate ribose 2060066.0000 3331700.0000 2528644.0000 688404.0000 1070490.0000 771052.0000 1742062.0000 3768828.0000 2591039.0000 Adenosine monophosphate 2066384.0000 21185746.0000 13023259.0000 2614458.0000 10968023.0000 2160267.0000 1395076.0000 Adenosine triphosphate 77767.0000 118322.0000 79953.0000 64779.0000 142307.0000 143008.0000 58647.0000 Adenylsuccinic acid 18303.0000 25836.0000 25276.0000 17501.0000 19224.0000 ADP 774099.0000 2036000.0000 1529042.0000 357555.0000 953806.0000 489327.0000 777981.0000 447184.0000 Allantoin 21116.0000 10120.0000 Aminoadipic acid 236509.0000 316976.0000 339762.0000 218790.0000 305292.0000 186762.0000 682960.0000 584821.0000 298790.0000 Argininosuccinic acid 15469.0000 Beta-Alanine Betaine 1557858.0000 3690359.0000 1927674.0000 2463429.0000 2706155.0000 2711772.0000 1692776.0000 1317169.0000 2682132.0000 Butyrylcarnitine 141893.0000 238428.0000 151479.0000 226972.0000 242034.0000 210855.0000 85599.0000 76419.0000 101867.0000 Carbamoyl phosphate Carnosine 47184.0000 48642.0000 39491.0000 55103.0000 35007.0000 CDP 23309.0000 62630.0000 85753.0000 51419.0000 Choline 160427952.0000 156923616.0000 145028944.0000 95457608.0000 141994912.0000 146104784.0000 158553632.0000 199977424.0000 138504064.0000 cis-Aconitic acid 741033.0000 988826.0000 777735.0000 633783.0000 854536.0000 467337.0000 1335604.0000 1274576.0000 1183327.0000 Citric acid 11466809.0000 14534911.0000 11923475.0000 6767400.0000 11052348.0000 5637846.0000 17491582.0000 19094722.0000 13614170.0000 Citrulline 168599.0000 258674.0000 156235.0000 483840.0000 277869.0000 384087.0000 Creatine 528820224.0000 646926592.0000 631926400.0000 468252896.0000 651075456.0000 500447680.0000 453487488.0000 480743040.0000 349356096.0000 Creatinine 1004231.0000 1930029.0000 1202486.0000 1042291.0000 1547767.0000 1043793.0000 748748.0000 779746.0000 1001837.0000 Cyclic AMP 19041.0000 21002.0000 21333.0000 18500.0000 18721.0000 25482.0000 30047.0000 23874.0000 Cytidine 863577.0000 529157.0000 601329.0000 844025.0000 714919.0000 1881253.0000 5041030.0000 5259692.0000 4353026.0000 Cytidine monophosphate 49343.0000 62329.0000 57036.0000 42075.0000 70488.0000 67824.0000 288529.0000 357590.0000 248955.0000 Cytidine triphosphate 13653.0000 19218.0000 Cytosine D-2-Hydroxyglutaric acid 956003.0000 1093854.0000 1052374.0000 1592446.0000 1265782.0000 1387705.0000 727102.0000 711674.0000 1185850.0000 D-Glucose 181976.0000 D-Glyceraldehyde 3-phosphate 3673881.0000 4539976.0000 3656681.0000 1038709.0000 8558430.0000 9064867.0000 7384047.0000 Dihydrofolic acid Dihydroxyacetone phosphate 5617455.0000 6661518.0000 5742942.0000 1117250.0000 3079494.0000 2018016.0000 7322530.0000 8398648.0000 7000460.0000 D-Ribose 5-phosphate 2624650.0000 2993512.0000 2427878.0000 1688940.0000 2507784.0000 2089485.0000 5994250.0000 4393680.0000 3677170.0000 D-Sedoheptulose 7-phosphate 916474.0000 975828.0000 871949.0000 846634.0000 885826.0000 829507.0000 1415715.0000 705932.0000 611627.0000 dTDP 19828.0000 14530.0000 FAD 79793.0000 76802.0000 86168.0000 64259.0000 75523.0000 73951.0000 93427.0000 85922.0000 80902.0000 Folic acid Fructose 1_6-bisphosphate 50094.0000 57563.0000 47174.0000 50472.0000 52432.0000 44709.0000 82345.0000 81597.0000 98337.0000 Fructose 6-phosphate 4300950.0000 4141307.0000 4003404.0000 2244772.0000 3316496.0000 2778046.0000 6329209.0000 5030416.0000 3145646.0000 Fumaric acid 2611044.0000 3373973.0000 3139174.0000 2543316.0000 3142902.0000 2806577.0000 2584478.0000 2785201.0000 3102560.0000 Glucose 6-phosphate 6483456.0000 7145008.0000 5807408.0000 3143759.0000 4034187.0000 3380600.0000 12371761.0000 8214848.0000 6976346.0000 Glutathione 31131498.0000 46888140.0000 36830164.0000 18196220.0000 35743596.0000 25057434.0000 55051464.0000 61515024.0000 39871216.0000 Glycerol 3-phosphate 1760922.0000 2595804.0000 1864260.0000 1159734.0000 1523641.0000 1164419.0000 3116034.0000 3212347.0000 2679002.0000 Glycine 3173883.0000 4069703.0000 3337115.0000 1740998.0000 2278392.0000 1933843.0000 1838709.0000 1839824.0000 1314362.0000 Guanidoacetic acid 22062670.0000 26373102.0000 21121166.0000 16695073.0000 19390628.0000 19525852.0000 5415031.0000 7332034.0000 7008316.0000 Guanine 12520082.0000 12730846.0000 12875113.0000 6609504.0000 10218157.0000 10400315.0000 23581570.0000 14956950.0000 10032831.0000 Guanosine 5061250.0000 3727541.0000 2981322.0000 1740189.0000 3407481.0000 4656076.0000 8389046.0000 7628702.0000 5490531.0000 Guanosine diphosphate 102777.0000 144581.0000 134213.0000 109202.0000 120639.0000 Guanosine monophosphate 379665.0000 1623922.0000 1325562.0000 207129.0000 651451.0000 238846.0000 Guanosine triphosphate Homocysteine Hypotaurine 1055750.0000 1375668.0000 1206216.0000 1117954.0000 1426690.0000 1096925.0000 642045.0000 600906.0000 405724.0000 Hypoxanthine 138581088.0000 152567632.0000 160403744.0000 107101528.0000 150444288.0000 125282288.0000 287922592.0000 210320192.0000 144897792.0000 Inosine 12947949.0000 12583922.0000 11853828.0000 8014814.0000 12131397.0000 11463682.0000 21276232.0000 24849668.0000 18186646.0000 Inosinic acid 29007.0000 20493.0000 67097.0000 87109.0000 23313.0000 L-Acetylcarnitine 2548570.0000 3371520.0000 2696500.0000 1440694.0000 2092861.0000 1619062.0000 2125837.0000 2042562.0000 1276838.0000 L-Alanine 17874268.0000 23496382.0000 20912010.0000 12803368.0000 17199000.0000 12738264.0000 13815673.0000 14867473.0000 11028115.0000 L-Arginine 35092384.0000 32286328.0000 L-Asparagine 1281641.0000 1282701.0000 1297822.0000 544382.0000 796307.0000 768044.0000 2646774.0000 2265530.0000 1574427.0000 L-Aspartic acid 31704584.0000 35755420.0000 34303628.0000 34150236.0000 38678300.0000 34678824.0000 23316784.0000 24153818.0000 22430486.0000 L-Carnitine 7737453.0000 8870147.0000 7645928.0000 5154351.0000 6911638.0000 5738016.0000 3754950.0000 2968219.0000 2563654.0000 L-Cystathionine 64483.0000 60288.0000 55614.0000 L-Cysteine 60910.0000 L-Cystine L-Glutamic acid 74643096.0000 100377976.0000 85221504.0000 83878152.0000 97591528.0000 91610784.0000 47168024.0000 52244588.0000 55887360.0000 L-Glutamine 36525804.0000 49104540.0000 44634320.0000 81412024.0000 83863928.0000 82084168.0000 32428016.0000 38391244.0000 53057388.0000 L-Histidine 16187126.0000 26243654.0000 17685852.0000 23020520.0000 24196060.0000 21256656.0000 20956392.0000 20221424.0000 16561519.0000 L-Isoleucine 22715286.0000 32908726.0000 26746630.0000 26178630.0000 34386128.0000 27787482.0000 25782560.0000 24581292.0000 20348858.0000 L-Kynurenine 340706.0000 553372.0000 422748.0000 441722.0000 702789.0000 465166.0000 318854.0000 321787.0000 224380.0000 L-Lactic acid 16691982.0000 27266880.0000 20264520.0000 9504299.0000 15021165.0000 9964841.0000 30550292.0000 29378334.0000 27061824.0000 L-Leucine 23285570.0000 36542104.0000 30436178.0000 30315646.0000 38340616.0000 30013018.0000 22348238.0000 21591120.0000 17879570.0000 L-Lysine 7588407.0000 9831838.0000 7464800.0000 6346726.0000 7924212.0000 6775733.0000 7716704.0000 L-Malic acid 25822192.0000 33592076.0000 31257638.0000 25472264.0000 30938176.0000 28205114.0000 26329604.0000 26952330.0000 30995240.0000 L-Methionine 13961598.0000 19296810.0000 16052837.0000 16052747.0000 21284316.0000 17797648.0000 19790974.0000 17468940.0000 12923680.0000 L-Phenylalanine 27478400.0000 37548508.0000 32651024.0000 34554320.0000 44086664.0000 34569652.0000 30933578.0000 28629976.0000 21465828.0000 L-Proline 252305952.0000 356229792.0000 302891328.0000 187594112.0000 256903984.0000 206058704.0000 133903096.0000 137854448.0000 108023264.0000 L-Serine 4325736.0000 5054681.0000 4313684.0000 2767444.0000 3421248.0000 2985537.0000 4336796.0000 3967708.0000 2958194.0000 L-Threonine 6783315.0000 14774755.0000 6988492.0000 6236523.0000 8919091.0000 7045572.0000 8169099.0000 7405829.0000 4615232.0000 L-Tryptophan 2400836.0000 3409567.0000 2823696.0000 2785217.0000 4315397.0000 3104772.0000 2084017.0000 1841516.0000 1491790.0000 L-Tyrosine 14076824.0000 18504580.0000 16929976.0000 17100720.0000 20665088.0000 16695232.0000 16045487.0000 14653861.0000 11949340.0000 L-Valine 20814550.0000 29023856.0000 24290426.0000 23986602.0000 29814698.0000 25124950.0000 19900872.0000 19727342.0000 15985299.0000 N6-Acetyl-L-lysine 48909.0000 N-Acetylglutamic acid 41805.0000 46807.0000 50880.0000 45409.0000 44857.0000 32775.0000 32383.0000 35934.0000 30941.0000 N-Acetylglutamine 16056.0000 23409.0000 16596.0000 31253.0000 31027.0000 35646.0000 22045.0000 19877.0000 24992.0000 N-Acetyl-L-alanine 182842.0000 224409.0000 173417.0000 208073.0000 258995.0000 188290.0000 172149.0000 157422.0000 104516.0000 N-Acetyl-L-methionine 53948.0000 59738.0000 56589.0000 34479.0000 42044.0000 44207.0000 149949.0000 101038.0000 101930.0000 N-Acetyl-L-phenylalanine N-Acetylornithine 54280.0000 N-Acetylserine 3094294.0000 3762100.0000 3861078.0000 1995970.0000 2777605.0000 2237815.0000 5692702.0000 5459863.0000 3838415.0000 NAD 265996.0000 382712.0000 297616.0000 459505.0000 497181.0000 282669.0000 319918.0000 392252.0000 331659.0000 NADH NADP 29509.0000 46640.0000 51678.0000 27157.0000 46655.0000 37357.0000 43111.0000 NADPH Niacinamide 250625456.0000 308701984.0000 286766720.0000 206342192.0000 287142848.0000 244091664.0000 347229856.0000 350834848.0000 288403744.0000 Nicotinic acid 75182.0000 O-Phosphoethanolamine 14903.0000 16142.0000 Ornithine 2455472.0000 3150409.0000 2415427.0000 2346514.0000 2715879.0000 1953390.0000 1907634.0000 Orotic acid 170953.0000 120572.0000 112244.0000 Orotidylic acid 11322.0000 11635.0000 Oxidized glutathione 3363520.0000 3479702.0000 4625505.0000 3489778.0000 2171040.0000 2707931.0000 4507516.0000 3157305.0000 3491972.0000 Oxoadipic acid Oxoglutaric acid 1203386.0000 1616960.0000 1376472.0000 1717199.0000 1971964.0000 1630728.0000 1190008.0000 1159800.0000 1256062.0000 Palmitic acid 77545256.0000 77731320.0000 52575008.0000 55504556.0000 30615868.0000 82202584.0000 39494224.0000 29333098.0000 52733928.0000 Pantothenic acid 3470762.0000 5620434.0000 4652011.0000 8385879.0000 9747636.0000 6869890.0000 3275910.0000 2683030.0000 2090463.0000 Phenol Phosphocreatine 303654.0000 397893.0000 237280.0000 294660.0000 312110.0000 284473.0000 179169.0000 170291.0000 118200.0000 Phosphoenolpyruvic acid 1519662.0000 2969651.0000 1955880.0000 1747525.0000 2772696.0000 1980353.0000 4670355.0000 3607868.0000 2726615.0000 Phosphoribosyl pyrophosphate 12355.0000 Phosphorylcholine 219125200.0000 354692352.0000 321084448.0000 501828352.0000 587945664.0000 472546944.0000 159917200.0000 237643776.0000 245089600.0000 Phosphoserine 26601.0000 30888.0000 22476.0000 28193.0000 Pipecolic acid Propionylcarnitine 628642.0000 761024.0000 634349.0000 277238.0000 354996.0000 282134.0000 132696.0000 143998.0000 125707.0000 Pyruvic acid 197706.0000 346057.0000 206587.0000 197081.0000 390708.0000 257380.0000 197301.0000 518029.0000 555064.0000 Ribose 1-phosphate 3236966.0000 4119786.0000 3256640.0000 2082295.0000 2991086.0000 2418602.0000 7404718.0000 5916298.0000 4617552.0000 Saccharopine S-Adenosylhomocysteine 124126.0000 147191.0000 124179.0000 94869.0000 116324.0000 120309.0000 261379.0000 270851.0000 181096.0000 S-Adenosylmethionine 3690742.0000 3955000.0000 2956480.0000 1995122.0000 3051240.0000 2682930.0000 1835589.0000 2459483.0000 1589878.0000 Sarcosine 15177809.0000 21971236.0000 17994794.0000 12044477.0000 15155810.0000 11826394.0000 12645599.0000 13800686.0000 9682115.0000 Succinic acid 786860.0000 1416852.0000 1076264.0000 839661.0000 1137196.0000 764058.0000 661768.0000 929513.0000 1021278.0000 Taurine 14902448.0000 17302700.0000 15692816.0000 22096378.0000 21625836.0000 21539238.0000 10031042.0000 9600713.0000 9143725.0000 Thymidine 34954.0000 51403.0000 25053.0000 Uracil 2258470.0000 2133860.0000 2150885.0000 3034173.0000 3856862.0000 3615646.0000 2347598.0000 1611006.0000 1193045.0000 Ureidopropionic acid 276402.0000 212663.0000 276597.0000 120218.0000 117966.0000 133379.0000 895690.0000 646616.0000 458531.0000 Ureidosuccinic acid 21929.0000 48601.0000 47983.0000 Uric acid Uridine 7488550.0000 7057661.0000 6395352.0000 9125715.0000 10826252.0000 11390328.0000 6092782.0000 5500702.0000 3910678.0000 Uridine 5^-diphosphate 2682907.0000 3516344.0000 2809604.0000 2461011.0000 3554893.0000 2437737.0000 1025025.0000 1355578.0000 945316.0000 Uridine 5^-monophosphate 3156559.0000 8725758.0000 7835432.0000 4522828.0000 6457858.0000 1995644.0000 847801.0000 Uridine diphosphate glucose 21345930.0000 23784430.0000 23961512.0000 22506890.0000 25838146.0000 22653530.0000 10457350.0000 13427980.0000 11990648.0000 Uridine diphosphate-N-acetylglucosamine 21593.0000 45185.0000 24408.0000 35661.0000 395427.0000 454768.0000 392858.0000 Uridine triphosphate 32117.0000 45085.0000 44936.0000 67456.0000 36031.0000 30015.0000 54544.0000 Valerylcarnitine 53405.0000 70476.0000 56433.0000 41723.0000 55306.0000 44009.0000 23098.0000 18943.0000 22544.0000 Xanthine 4581563.0000 4833809.0000 4553732.0000 2092104.0000 3457150.0000 3074988.0000 5354108.0000 4029812.0000 2284080.0000 Xanthosine Xanthylic acid MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant 1-Methylnicotinamide 19.01 137.0709 2,3-Diphosphoglyceric acid 14.96 264.952 3-Hydroxybutyric acid 7.44 103.0401 3-Phosphoglyceric acid 14.6 184.9857 4,5-Dihydroorotic acid 8.64 157.0255 4-Hydroxyproline 10.86 132.0655 5'-Methylthioadenosine 4.09 298.0968 5-Phosphoribosylamine 12.5 228.0279 6-Phosphogluconic acid 15.79 275.0174 Acetoacetic acid 10.32 101.0244 Acetyl-CoA 10.84 810.1331 Acetylcysteine 7.19 164.0376 Adenine 6.21 136.0618 Adenosine 5.85 268.104 Adenosine diphosphate ribose 12.17 558.0644 Adenosine monophosphate 11.08 348.0704 Adenosine triphosphate 14.45 505.9885 Adenylsuccinic acid 16.07 462.0668 ADP 12.55 426.0221 Allantoin 10.54 157.0367 Aminoadipic acid 7.05 160.0615 Argininosuccinic acid 13.6 289.1154 Beta-Alanine 10.43 90.055 Betaine 7.86 118.0863 Butyrylcarnitine 5.47 232.1543 Carbamoyl phosphate 27.03 139.9754 Carnosine 12.03 227.1139 CDP 14.62 404.0255 Choline 14.85 104.107 cis-Aconitic acid 15.6 173.0092 Citric acid 15.99 191.0197 Citrulline 12.3 176.103 Creatine 11.03 132.0768 Creatinine 6.47 114.0662 Cyclic AMP 6.95 328.0452 Cytidine 8.45 244.0928 Cytidine monophosphate 12.65 324.0591 Cytidine triphosphate 16.76 481.9772 Cytosine 9.17 112.0505 D-2-Hydroxyglutaric acid 12.7 147.0299 D-Glucose 10.43 179.0561 D-Glyceraldehyde 3-phosphate 13.57 168.9908 Dihydrofolic acid 14.97 444.1626 Dihydroxyacetone phosphate 12.7 168.9908 D-Ribose 5-phosphate 12.4 229.0119 D-Sedoheptulose 7-phosphate 13.54 289.033 dTDP 12.84 401.0157 FAD 8.91 786.1644 Folic acid 14.44 442.147 Fructose 1,6-bisphosphate 14.21 338.9888 Fructose 6-phosphate 13.29 259.0224 Fumaric acid 13.53 115.0037 Glucose 6-phosphate 14.16 259.0224 Glutathione 11.58 308.0911 Glycerol 3-phosphate 12.28 171.0064 Glycine 12.07 76.0393 Guanidoacetic acid 12.07 118.0611 Guanine 8.73 152.0567 Guanosine 9.09 284.0989 Guanosine diphosphate 15.63 442.0171 Guanosine monophosphate 13.99 364.0653 Guanosine triphosphate 16 521.9834 Homocysteine 8.56 134.0281 Hypotaurine 11.44 110.027 Hypoxanthine 6.97 137.0458 Inosine 7.72 269.088 Inosinic acid 12.77 349.0544 L-Acetylcarnitine 7.55 204.123 L-Alanine 11.19 90.055 L-Arginine 20.26 175.119 L-Asparagine 11.76 133.0608 L-Aspartic acid 12.12 132.0302 L-Carnitine 9.72 162.1125 L-Cystathionine 13.7 223.0747 L-Cysteine 14.89 122.027 L-Cystine 13.13 239.0166 L-Glutamic acid 11.85 148.0604 L-Glutamine 11.54 147.0764 L-Histidine 11.08 156.0768 L-Isoleucine 7.39 132.1019 L-Kynurenine 7.39 209.0921 L-Lactic acid 7.22 89.0244 L-Leucine 7.83 132.1019 L-Lysine 19.26 147.1128 L-Malic acid 13.55 133.0142 L-Methionine 8.12 150.0583 L-Phenylalanine 6.84 166.0863 L-Proline 9.27 116.0706 L-Serine 12.21 106.0499 L-Threonine 10.94 118.051 L-Tryptophan 8.15 205.0972 L-Tyrosine 9.54 182.0812 L-Valine 8.98 118.0863 N6-Acetyl-L-lysine 3.3 189.1234 N-Acetylglutamic acid 11.69 190.071 N-Acetylglutamine 7.97 187.0724 N-Acetyl-L-alanine 6.84 132.0655 N-Acetyl-L-methionine 4.06 192.0689 N-Acetyl-L-phenylalanine 2.83 208.0968 N-Acetylornithine 10.01 175.1077 N-Acetylserine 8.23 146.0459 NAD 11.09 664.1164 NADH 10.72 664.1175 NADP 14.18 744.0827 NADPH 14.22 744.0838 Niacinamide 4.43 123.0553 Nicotinic acid 6.75 122.0248 O-Phosphoethanolamine 13.21 142.0264 Ornithine 17.83 133.0972 Orotic acid 7.76 155.0098 Orotidylic acid 11.51 369.033 Oxidized glutathione 14.85 613.1592 Oxoadipic acid 9.52 159.0299 Oxoglutaric acid 13.15 145.0142 Palmitic acid 2.62 255.233 Pantothenic acid 6.24 220.118 Phenol 3.37 93.0346 Phosphocreatine 12.55 210.0285 Phosphoenolpyruvic acid 15.11 166.9751 Phosphoribosyl pyrophosphate 15.44 388.9445 Phosphorylcholine 12.06 184.0733 Phosphoserine 11.87 186.0162 Pipecolic acid 8.79 130.0863 Propionylcarnitine 6.4 218.1387 Pyruvic acid 6.05 87.0088 Ribose 1-phosphate 12.36 229.0119 Saccharopine 11.81 275.1249 S-Adenosylhomocysteine 9.96 383.1143 S-Adenosylmethionine 12.34 399.1445 Sarcosine 11.21 90.055 Succinic acid 12.7 117.0193 Taurine 11.66 124.0074 Thymidine 4.47 241.083 Uracil 6.72 113.0346 Ureidopropionic acid 11.73 131.0462 Ureidosuccinic acid 14.2 175.036 Uric acid 8.95 169.0356 Uridine 6.72 243.0623 Uridine 5'-diphosphate 13.79 405.0095 Uridine 5'-monophosphate 12.46 323.0286 Uridine diphosphate glucose 13.8 565.0478 Uridine diphosphate-N-acetylglucosamine 12.57 608.0889 Uridine triphosphate 15.77 482.9613 Valerylcarnitine 4.98 246.17 Xanthine 8.66 151.0261 Xanthosine 7.88 283.0684 Xanthylic acid 12.64 363.0347 METABOLITES_END #END