#METABOLOMICS WORKBENCH leilirohani_20200508_183528 DATATRACK_ID:2003 STUDY_ID:ST001377 ANALYSIS_ID:AN002297 PROJECT_ID:PR000942 VERSION 1 CREATED_ON May 13, 2020, 9:40 am #PROJECT PR:PROJECT_TITLE Inferring metabolism dynamics between naïve and primed human pluripotent stem PR:PROJECT_TITLE cells in the stirred suspension bioreactor PR:PROJECT_TYPE Targeted and untargeted MS PR:PROJECT_SUMMARY We used high-resolution LC-MS targeted metabolomics to screen for extracellular PR:PROJECT_SUMMARY metabolites in the media of bioreactor-cultured, naïve and primed human PR:PROJECT_SUMMARY pluripotent stem cells. PR:INSTITUTE University of Calgary PR:DEPARTMENT Biochemistry and Molecular Biology PR:LABORATORY Stem Cell Research PR:LAST_NAME Rohani PR:FIRST_NAME Leili PR:ADDRESS 405J, 1919 University Drive, NW PR:EMAIL leili.rohanisarvesta@ucalgary.ca PR:PHONE 5879681647 #STUDY ST:STUDY_TITLE Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent ST:STUDY_TITLE stem cells (hPSCs) ST:STUDY_SUMMARY Although cell therapies require large numbers of quality-controlled hPSCs, ST:STUDY_SUMMARY existing technologies are limited in their ability to efficiently grow and scale ST:STUDY_SUMMARY stem cells. We report here that cell-state conversion from primed-to-naïve ST:STUDY_SUMMARY pluripotency enhances the biomanufacturing of hPSCs. Naïve hPSCs exhibit ST:STUDY_SUMMARY superior growth kinetics and aggregate formation characteristics in stirred ST:STUDY_SUMMARY suspension bioreactors compared to their primed counterparts. Moreover, we ST:STUDY_SUMMARY demonstrate the role of the bioreactor mechanical environment in the maintenance ST:STUDY_SUMMARY of naïve pluripotency, through transcriptomic enrichment of mechano-sensing ST:STUDY_SUMMARY signaling for cells in the bioreactor along with a decrease in expression of ST:STUDY_SUMMARY lineage-specific and primed pluripotency hallmarks. Bioreactor-cultured, naïve ST:STUDY_SUMMARY hPSCs express epigenetic regulatory transcripts associated with naïve ST:STUDY_SUMMARY pluripotency, and display hallmarks of X-chromosome reactivation. They exhibit ST:STUDY_SUMMARY robust production of naïve pluripotency metabolites and display reduced ST:STUDY_SUMMARY expression of primed pluripotency cell surface markers. We also show that these ST:STUDY_SUMMARY cells retain the ability to undergo targeted differentiation into beating ST:STUDY_SUMMARY cardiomyocytes, hepatocytes, and neural rosettes. They additionally display ST:STUDY_SUMMARY faster kinetics of teratoma formation compared to their primed counterparts. ST:STUDY_SUMMARY Naïve bioreactor hPSCs also retain structurally stable chromosomes. Our ST:STUDY_SUMMARY research corroborates that converting hPSCs to the naïve state enhances hPSC ST:STUDY_SUMMARY manufacturing and indicates a potentially important role for the bioreactor’s ST:STUDY_SUMMARY mechanical environment in maintaining naïve pluripotency. ST:INSTITUTE University of Calgary ST:DEPARTMENT Biochemistry and Molecular Biology ST:LABORATORY Stem Cell Research ST:LAST_NAME Rohani ST:FIRST_NAME Leili ST:ADDRESS 405J, 1919 University Drive, NW ST:EMAIL leili.rohanisarvesta@ucalgary.ca ST:PHONE +1 5879681647 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Blank_mid6 Cell line:none | Status:none RAW_FILE_NAME=2019_10_07LR_RPIP15_Blank_mid6.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Blank_mid7 Cell line:none | Status:none RAW_FILE_NAME=2019_10_07LR_RPIP15_Blank_mid7.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Blank_mid8 Cell line:none | Status:none RAW_FILE_NAME=2019_10_07LR_RPIP15_Blank_mid8.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Blank_mid9 Cell line:none | Status:none RAW_FILE_NAME=2019_10_07LR_RPIP15_Blank_mid9.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Blank_mid10 Cell line:none | Status:none RAW_FILE_NAME=2019_10_07LR_RPIP15_Blank_mid10.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M1_1 Cell line:H1 | Status:day 1 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M1_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M1_2 Cell line:H1 | Status:day 2 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M1_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M2_1 Cell line:H1 | Status:day 3 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M2_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M2_2 Cell line:H1 | Status:day 4 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M2_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M3_1 Cell line:H1 | Status:day 3 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M3_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M3_2 Cell line:H1 | Status:day 3 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M3_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M4_1 Cell line:H1 | Status:day 4 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M4_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M4_2 Cell line:H1 | Status:day 4 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M4_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M5_1 Cell line:H1 | Status:day 5 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M5_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H1-M5_2 Cell line:H1 | Status:day 5 RAW_FILE_NAME=2019_10_07LR_RPIP15_H1_M5_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H9-naive-bioreactor-1- Cell line:H9 | Status:naive RAW_FILE_NAME=2019_10_07LR_RPIP15_H9_naive_bioreactor_1_.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H9-naive-bioreactor-2 Cell line:H9 | Status:naive RAW_FILE_NAME=2019_10_07LR_RPIP15_H9_naive_bioreactor_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H9-naive-bioreactor-3 Cell line:H9 | Status:naive RAW_FILE_NAME=2019_10_07LR_RPIP15_H9_naive_bioreactor_3.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H9-primed-bioreactor-1 Cell line:H9 | Status:primed RAW_FILE_NAME=2019_10_07LR_RPIP15_H9_primed_bioreactor_1_.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H9-primed-bioreactor-2 Cell line:H9 | Status:primed RAW_FILE_NAME=2019_10_07LR_RPIP15_H9_primed_bioreactor_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_H9-primed-bioreactor-3 Cell line:H9 | Status:primed RAW_FILE_NAME=2019_10_07LR_RPIP15_H9_primed_bioreactor_3.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M1_1 Cell line:H1 | Status:day 1 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M1_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M1_2 Cell line:H1 | Status:day 2 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M1_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M2_1 Cell line:H1 | Status:day 3 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M2_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M2_2 Cell line:H1 | Status:day 4 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M2_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M3_1 Cell line:H1 | Status:day 3 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M3_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M3_2 Cell line:H1 | Status:day 3 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M3_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M4_1 Cell line:H1 | Status:day 4 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M4_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M4_2 Cell line:H1 | Status:day 4 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M4_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M5_1 Cell line:H1 | Status:day 5 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M5_1.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H1-M5_2 Cell line:H1 | Status:day 5 RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H1_M5_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-1 Cell line:H9 | Status:naive RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H9_naive_bioreactor_1_.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-2 Cell line:H9 | Status:naive RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H9_naive_bioreactor_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-3 Cell line:H9 | Status:naive RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H9_naive_bioreactor_3.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-1 Cell line:H9 | Status:primed RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H9_primed_bioreactor_1_.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-2 Cell line:H9 | Status:primed RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H9_primed_bioreactor_2.raw SUBJECT_SAMPLE_FACTORS - 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-3 Cell line:H9 | Status:primed RAW_FILE_NAME=2019_10_07LR_RPIP15_Ref_Media_H9_primed_bioreactor_3.raw #COLLECTION CO:COLLECTION_SUMMARY Extracellular metabolite dynamics was analyzed during primed to naïve CO:COLLECTION_SUMMARY conversion in the bioreactor. For this, the media of H1 hESCs in the bioreactor CO:COLLECTION_SUMMARY was gradually switched from mTESR1 to RSeT media within 6 days of culture (D2 to CO:COLLECTION_SUMMARY D6) and the media was collected every day for the analysis. For H9 hESCs, the CO:COLLECTION_SUMMARY extracellular metabolites were analyzed in the spent media of primed and CO:COLLECTION_SUMMARY established naïve (P5) hPSCs in the bioreactor, and the media of each primed or CO:COLLECTION_SUMMARY naïve hPSCs was collected at day four of bioreactor culture. CO:SAMPLE_TYPE Stem cells #TREATMENT TR:TREATMENT_SUMMARY For H1 hESCs, the extracellular metabolite dynamics was analyzed during primed TR:TREATMENT_SUMMARY to naïve conversion in the bioreactor. For this, the media of H1 hESCs in the TR:TREATMENT_SUMMARY bioreactor was gradually switched from mTESR1 to RSeT media within 6 days of TR:TREATMENT_SUMMARY culture (D2 to D6) and the media was collected every day for the analysis. For TR:TREATMENT_SUMMARY H9 hESCs, the extracellular metabolites were analyzed in the spent media of TR:TREATMENT_SUMMARY primed and established naïve (P5) hPSCs in the bioreactor, and the media of TR:TREATMENT_SUMMARY each primed or naïve hPSCs was collected at day four of bioreactor culture. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The metabolite extraction was started using LC-MS or HPLC grade methanol SP:SAMPLEPREP_SUMMARY (Sigma-Aldrich, 1.06035). Briefly, a 950 μL of pre-chilled 50% MeOH/H2O was SP:SAMPLEPREP_SUMMARY added to a 50 μL of the bioreactor-collected media (making a D20 dilution), and SP:SAMPLEPREP_SUMMARY the samples were incubated on ice for 30 min to allow for full extraction. SP:SAMPLEPREP_SUMMARY Macromolecules were then pelleted by centrifugation at max speed (~18,000-21,000 SP:SAMPLEPREP_SUMMARY g) for 10 mins in a bench top centrifuge (preferably chilled) to extract the SP:SAMPLEPREP_SUMMARY supernatant. Further, the extracted samples were stored in -80 prior to running SP:SAMPLEPREP_SUMMARY HPLC-MS. SP:PROCESSING_STORAGE_CONDITIONS Room temperature SP:EXTRACTION_METHOD 50% Methanol in water SP:EXTRACT_STORAGE On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Agilent Zorbax RRHT SB-C18 (50 x 2.1 mm, 1.8 um) CH:FLOW_RATE 0.6 ml/min CH:SOLVENT_A 10mM tributylamine, 10mM acetate pH 7.5 in 97/3% (v/v) MS grade water/methanol CH:SOLVENT_B MS grade acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Ian Lewis AN:OPERATOR_NAME Ryan Groves #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The mass spectrometer was run in negative full scan mode at a resolution of MS:MS_COMMENTS 140,000 scanning from 50-750m/z. Data was processed using MAVEN. MS:CAPILLARY_TEMPERATURE 275 MS:SPRAY_VOLTAGE 2.50 kV #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS relative signal intensity compared to media only MS_METABOLITE_DATA_START Samples 2019_09_13_RPIP15_Blank_mid6 2019_09_13_RPIP15_Blank_mid7 2019_09_13_RPIP15_Blank_mid8 2019_09_13_RPIP15_Blank_mid9 2019_09_13_RPIP15_Blank_mid10 2019_09_13_RPIP15_H1-M1_1 2019_09_13_RPIP15_H1-M1_2 2019_09_13_RPIP15_H1-M2_1 2019_09_13_RPIP15_H1-M2_2 2019_09_13_RPIP15_H1-M3_1 2019_09_13_RPIP15_H1-M3_2 2019_09_13_RPIP15_H1-M4_1 2019_09_13_RPIP15_H1-M4_2 2019_09_13_RPIP15_H1-M5_1 2019_09_13_RPIP15_H1-M5_2 2019_09_13_RPIP15_H9-naive-bioreactor-1- 2019_09_13_RPIP15_H9-naive-bioreactor-2 2019_09_13_RPIP15_H9-naive-bioreactor-3 2019_09_13_RPIP15_H9-primed-bioreactor-1 2019_09_13_RPIP15_H9-primed-bioreactor-2 2019_09_13_RPIP15_H9-primed-bioreactor-3 2019_09_13_RPIP15_Ref-Media-H1-M1_1 2019_09_13_RPIP15_Ref-Media-H1-M1_2 2019_09_13_RPIP15_Ref-Media-H1-M2_1 2019_09_13_RPIP15_Ref-Media-H1-M2_2 2019_09_13_RPIP15_Ref-Media-H1-M3_1 2019_09_13_RPIP15_Ref-Media-H1-M3_2 2019_09_13_RPIP15_Ref-Media-H1-M4_1 2019_09_13_RPIP15_Ref-Media-H1-M4_2 2019_09_13_RPIP15_Ref-Media-H1-M5_1 2019_09_13_RPIP15_Ref-Media-H1-M5_2 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-1 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-2 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-3 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-1 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-2 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-3 Factors Cell line:none | Status:none Cell line:none | Status:none Cell line:none | Status:none Cell line:none | Status:none Cell line:none | Status:none Cell line:H1 | Status:day 1 Cell line:H1 | Status:day 2 Cell line:H1 | Status:day 3 Cell line:H1 | Status:day 4 Cell line:H1 | Status:day 3 Cell line:H1 | Status:day 3 Cell line:H1 | Status:day 4 Cell line:H1 | Status:day 4 Cell line:H1 | Status:day 5 Cell line:H1 | Status:day 5 Cell line:H9 | Status:naive Cell line:H9 | Status:naive Cell line:H9 | Status:naive Cell line:H9 | Status:primed Cell line:H9 | Status:primed Cell line:H9 | Status:primed Cell line:H1 | Status:day 1 Cell line:H1 | Status:day 2 Cell line:H1 | Status:day 3 Cell line:H1 | Status:day 4 Cell line:H1 | Status:day 3 Cell line:H1 | Status:day 3 Cell line:H1 | Status:day 4 Cell line:H1 | Status:day 4 Cell line:H1 | Status:day 5 Cell line:H1 | Status:day 5 Cell line:H9 | Status:naive Cell line:H9 | Status:naive Cell line:H9 | Status:naive Cell line:H9 | Status:primed Cell line:H9 | Status:primed Cell line:H9 | Status:primed Pyruvate 19130.54 13694.65 15617.58 9440.81 14266.6 182114.83 153551.5 175914.45 142518.55 183612.69 163362.08 204710.05 207015.23 270089.25 204316.86 103718.54 189152.64 138419.66 99691.62 139186.45 115717.85 145343.23 127989.41 181817.05 180480.73 173681.59 168964.22 218358.3 203979.38 274755.53 237808.42 260657.75 290514.28 228701.52 225358.33 190077.12 195382.58 L-Alanine 2450.7 3066.22 3293.82 4731.79 2575.05 339421.34 236446.05 519150.84 473889.38 780970 903638.5 1470821.12 1370521.62 1947808.5 1957785.38 2113074.75 1946130.12 2243909.25 374749.91 357588.28 472814.38 47140.23 73707.6 106757.62 73674.2 76306.73 82949.48 91190.56 101064.65 92815.15 79686.85 67422.3 79448.29 56659.71 86660.25 84800.91 75427.91 lactate 35038.45 34709.1 54222.32 38128.69 59305.89 39271008 35160632 16831590 18496208 13251512 13463163 12882235 13242920 9562433 9238140 80045408 75418624 70192648 63984236 73691592 74447072 48745.69 45999.29 61669.03 42306.02 29415.42 34979.46 31395.12 31369.03 27680.12 25902 29108.72 32085.23 21361.55 60644.89 42148.88 70452.95 Acetoacetate 3908.11 7455.8 9128.84 3862.59 5712.08 1615662 1348549.5 1963866.38 1571996 1959971.12 1897742.88 2589767.25 1930904.62 3137717.75 3211359.25 1639112.5 1783902.38 1759722.12 764795.56 1007625.69 1149230 1660988 1512716 2075122.88 1671743.88 2218103.25 1839119.38 2642084 2324332.25 2498177.25 2327350.25 2935162.75 3037026 3014187 2088448 1949001 2153431.75 L-Serine - - - - - 289708.41 275413.69 329299.5 290979.53 360013.34 369005.59 500931.41 450067.09 628127.81 545649.75 389498.38 377351.25 360384.5 256388.12 221676.25 298540.16 281464.78 295415.91 380613.75 336548.16 366503.5 342688.88 476457.03 544730.38 585021.19 492475.38 488789.16 546507.19 434227.38 329315 331335.31 380855.91 MALEAMATE - - - 1200.14 - - - - - - - - 3900.12 6842.18 - 8989.46 - 5165.68 21590.35 39417.12 25662.87 3057.55 4617.41 - 1850.17 - - - - 4687.91 - 46883.76 65169.69 63863.64 52914.14 57036.11 56540.7 L-PROLINE 1760.52 2414.14 2111.47 1633.67 1957.94 444008.62 373576.88 363436.16 250778.61 296779.38 348126.12 279710.59 396049.97 373744.78 322141.34 413968.53 443536.16 463444.84 479165.66 524793.94 534569.38 287798.62 239950.81 338303.09 301440.84 295025 253013.38 337405.47 360030.91 304356.84 260651.23 275052.53 318393.16 263837.53 323910.28 314441.12 406261.34 3-Methyl-2-oxobutanoic acid 130635.37 110114.27 121636.31 166067.42 129996.62 578685.12 515588.03 333186.72 285483.16 262138.62 234617.19 188793.19 165824.58 153209.52 160398.66 539743.88 516072.03 484413.16 665540.69 602439.5 627783.06 131475.08 120442.51 152793.52 141153.3 102738.41 125126.25 146808.92 116460.38 145305.27 176199.02 125508.5 117481.52 134379.48 133460.73 144632.08 134640.52 Fumarate 155231.58 178497.58 143351.28 148157.8 166806.48 45667.03 57368.36 41439.5 42905.88 47972.18 45563.57 43410.61 40748.93 38510.64 58504.75 48289.69 58613.14 60212.42 54436.55 73457.59 58768.08 52754.71 54373.72 46544.36 49256.23 59138.2 77863.44 30515.99 53788.67 63658.14 63359.45 48943.82 26202.96 51308.54 47956.86 83758.02 39114.39 L-Valine 4170.12 6956.04 6295.66 4456.77 7272.61 859716 1054967.88 952495.19 1060229.5 1310949 1627942.38 1742398 1678923.5 2317524.25 2192872 2033723.62 1811228.38 2086597.12 1010601.31 873803.81 1040039.19 879564.06 911877.81 1297077 1190414 1371562.12 1315609.12 1784044.38 1842970.5 1858422 1723391.62 1769700 1794101.62 1979473 1270628 1159016 1130607.62 Succinate 588461.25 554479.25 591843.81 634912.69 683580.31 228274.3 198275.83 152032.64 148488.92 157764.3 183955.8 229268.64 216194.62 345259.22 282181.16 741049.69 681969.06 659760.69 271586.94 223916.27 316427.5 136527.02 135451.33 153265.72 158450.75 157331.22 147371.31 117852.52 136256.09 128514.4 169901.27 146866.94 151103.58 118367.71 137249.03 149942.98 110164.21 L-Threonine - - - - - 642443.44 647389.31 794553.75 635429.25 957953.31 991489.94 1043902.44 1252785.38 1681741 1410001.38 1219223.62 1339526.75 1230239 585445.94 541132.56 718865.44 575528.19 525166.62 875582.69 720344.94 926060.81 720643.94 1255015.38 1328702.75 1443650.12 1138064.12 1178013.5 1354626.12 1280372 770240.31 713497.69 703267.81 4-HYDROXYBENZALDEHYDE 12120341 12338707 12830395 13172663 14162775 13499464 13108793 14241696 14286904 13914341 13257128 13689539 13524573 14883825 13390832 12405696 12139328 11430955 11671561 11922563 13658455 12699344 12555863 14043607 13622575 13989115 13894960 13784745 14522739 13845429 13534291 11942657 11649272 12488387 12788421 13641755 14212283 Thymine 3369 - - 1791.03 1788.13 583644.75 607260.38 69761.93 429186.38 289642.84 283436.88 119304.37 133684.22 11577.96 6122.12 4219.23 11043.99 12551.91 530139 508099.25 724422.06 - 506250.25 131209.7 509322.09 297493.34 292462.88 173935.69 138715.23 2302.04 4948.16 - - 1105.43 590889.19 589425.69 611071.81 Pyroglutamic acid 2721.91 1600.42 4558.16 2062.91 4857.51 8991.94 16827.89 11279.57 14374.27 9955.89 11476.03 12263.85 7064.14 11516.16 12355.67 21387.01 25073.22 17206.56 12488.62 23841.8 20555.47 15572.47 16403.72 10329.21 10238.67 8005.33 10988.57 12539.84 4665.63 8170.96 8946.9 2342.03 5324.6 2555.92 16662.88 18981.89 14629.24 5-Oxoproline 2279.23 2697.55 4935.87 2300.71 5141.81 165360.61 164433.08 95637.29 97324.23 82619.04 90614.6 85132.52 81205.66 90781.37 84082.52 361736.25 330745.47 302744.59 215545.44 243734.56 238194.92 84133.88 77977.8 65388.78 64031.54 61305.64 56948.7 75506.84 50612.06 60988.22 45390.56 61950.74 74593.43 74404.28 95878.31 82479.35 92377.37 Citraconic acid 126157.5 125327.9 143049.16 135090.06 135094.45 109904.97 160726.75 153945.23 137582.16 163494.67 176058.62 216717.67 194295.39 315624.06 316115.12 263399.66 281743.59 284503.41 128148.22 194243.27 137253.55 189206.89 137432.58 134819.86 166869.12 206342.06 196688.56 226752.73 228735.14 301750.22 315966.72 333727.38 307520.75 300410.53 120409.9 237570.33 137085.59 L-Leucine 22788.87 27410.22 28110.35 22137.62 20730 3907600.25 3292977.25 4464197.5 4419452 5690724.5 5933530.5 7136097.5 7047806.5 9739145 9656058 8958039 8771198 7914408.5 3648967.75 4182994.75 3863053.25 4464524.5 4364420.5 5238214.5 5566841.5 6226406.5 6175772 8330020 7125594.5 8103873.5 7754442.5 8154158.5 8137057.5 8086156.5 5236612.5 4714482 4741368.5 L-Asparagine 2328.29 2012.68 - 2294.61 2002.48 284950.72 251937.2 270066.66 210042.39 228484.42 247931.05 217860.02 253162.33 293034.19 282689.5 338935.75 371240.22 304394.97 286679.34 249174.23 332112.59 203890.27 202035.31 239558.88 192488.3 241143.52 173546.06 262811.28 257716.81 240858.31 214019.62 213889.5 218511.8 216647.98 274230.22 221479.2 230654.88 L-ORNITHINE - 1300.18 2135.8 - - 81089.15 86798.3 54944.57 72116.3 73741.69 55272.69 70292.91 70789.48 84420.59 68059.34 68226.81 - 52379.98 88106.09 84652.22 95739.75 54419.34 55020.22 54562.27 60818.09 62439.77 57651.97 73298.24 57561.68 57592.88 62243.34 53204.34 55672.58 49972.29 52644.57 49100.59 59674.06 Aspartate - 2016.62 2025.64 - - 350842.16 348376.66 267880.09 237080.38 268423.09 254729.48 242683 212994.77 280348.47 225220.08 6352.02 5248.07 5692.02 400749.78 424217.25 443383.09 343320.38 329101.09 268446.16 288847.75 295190.59 297889.5 349199.78 227065.27 276014.25 272578.56 282622.5 292497.28 335735.31 466655.38 354328.41 412604.78 malate 73831.29 54473.78 75204.91 76853.24 60526.93 279256.94 269933.5 159854.88 150569.17 139375.92 99772.63 115792.05 111099.78 73135.44 104770.29 463906.78 408498.75 351272.84 311322.84 305654.09 329612.16 82779.98 63225.16 112557.54 74500.06 95258.72 85151.05 70157.07 72001.55 86618.6 78552.92 85147.57 71979.91 83586.94 86045.06 73687.32 89975.37 Hypoxanthine - - - - - 49933.64 66239.38 55692.74 56529.19 50051.97 49480.44 52261.52 50534.45 69465.09 67296.71 2578.69 2989.32 - 13659.71 16747.14 19357.93 79111.69 95946.6 63244.7 80820.12 75321.59 82894.98 90466.6 69434.56 62624.11 59856.84 54618.19 54776.25 49279.8 121752.84 110946.5 112827.06 a-ketoglutarate 66472.27 83542.57 70899.87 76586.8 77362.57 304476.31 95719.4 419065.12 385510.41 744191 626414.31 1283960 1325876.38 2108537.25 1902268.88 4370222 4358241.5 4169670.75 92627.69 110402.71 118901.12 75435.04 70442.02 148723.94 148668.34 219014.06 228684.88 416974.84 380866.84 417328.09 441776.16 602261.19 601808.94 558794.69 85523.28 66322.54 71416.73 L-Glutamine - - - - - 4323567.5 4469475.5 5799356 5658594 6525652 6094453.5 7753528 7543737.5 8160076.5 7303309.5 5385495.5 5082175.5 5155346.5 3564248.25 3643408.75 4003424 5981808.5 5963541.5 4970694 4557390.5 4284458.5 3693598.75 3762125.75 3693262.75 2618275.25 2132839.75 1948623.38 2118025 1973651.12 6150644 5996224 5968493.5 L-LYSINE - - - - - 158099.89 138373.73 175342.08 185701.61 200981.45 237968.25 271787.53 289251.12 408667 393733.38 287043.16 261046.8 280536.59 98017.68 126136.41 135033.55 140740.17 115632.85 197548.2 220369.25 228851.08 228215.42 314148.78 285481.03 427815.5 300079.41 311433.19 241840.92 230745.83 135845.3 153579.16 145787.05 L-Glutamate - - - - - 1281139.88 1296751.62 738412.5 772735.94 649432.38 633733.75 666581.12 661634.19 840398.31 705659.5 2324368 2211544.75 1957706.62 1365826.25 1443171.5 1413685.62 507063.25 512407.62 499872.25 471684.16 561597 417668.25 565885.69 423865.84 425985.5 421246.12 356678.09 429489.75 440719.03 638680.88 553971.06 625487.44 CITRAMALATE 45603.74 47739.92 55443.51 43812.34 40555.04 71316.07 54732.2 173086.38 155317.27 418849.75 353425.66 611960.25 502383.16 897243.81 914823.81 752391.75 868724.19 693905.88 67507.51 58699.31 84642.1 57547.12 41442.65 213646.61 193688.27 417729.34 458092.97 795963 603164.31 837821.19 780904.56 851473.94 917998.19 1007777.81 69444.34 55919.21 57560.39 L-METHIONINE - - - - - 286431.78 275194.97 335814.28 292412.72 384550.38 348618.91 476080.5 446950.12 689392.81 515463.91 730902.31 685764.69 602135.5 250155.23 263911.34 264803.75 263541.53 257566.2 389739.97 297395.19 420764.66 375177.84 524215.03 482499.66 487789.91 441729.09 569597.06 582131.81 599371.81 421980.53 359714.59 329722.78 D-Ribose - - 2053.01 1993.34 - 315918.22 257606 295006.59 346761.19 301888.91 390638.09 361411.66 473296.59 496896.16 537915.5 367687.5 326818.16 348849.09 193645.67 213549.95 211118.06 269555.5 300980.84 349158.28 292936.97 333257.28 358308.03 516141.72 460834.84 537534.75 409659.25 508513.59 601256 481785.75 342813.41 347406.16 391417.91 L-HISTIDINE - - 1534.01 - - 149887.89 134753.44 166616.41 122165.65 178976.62 166934.58 187545.89 181554.98 248153.3 181314.45 295310.12 265208.03 236951.95 143156.45 194678 150681.39 153243.95 124527.94 157272.56 142177.22 174409.58 135323.7 196417.23 137475.81 197155.14 156308.81 231407.39 253607.73 235015.75 188961.8 171120.7 173221.95 3-HYDROXY-3-METHYLGLUTARATE 4078.8 6469.26 3983.17 7980.8 6746.25 2230748.75 1927511.62 2690037.75 2333885.25 2896827.25 2723695 3466655.25 3154147.25 4732692 4305195 2600344.25 2543730.25 2205262.75 1214470.5 1535843 1478572.5 2540090.25 2153908.25 2833253.75 2006453.38 2875444 2635042.5 3801537.25 3686770 3970222.75 3670132.75 4489255.5 4617478.5 5054112.5 2874982.25 2360998.75 3103778 L-Phenylalanine 1639.75 1883.46 1814.51 1814.96 - 396787.75 314586.5 385426.91 362564.34 501689.03 477918.34 577197.19 559066.44 867011.94 739810.19 842985.19 690500.75 739564.25 347526.72 429783 405396.5 320121.5 329886.66 419875.5 385798.88 427455 496825.47 632318.06 542157.31 611350.25 588478.25 612752.31 675043.31 740822.25 465877.75 435635.59 403625.16 arginine - 1668.73 - - - 253633 235707.92 316862.97 333160.56 326989.34 347919.5 339956.47 325098.16 469464.12 447457.75 351328.03 365049.91 301246.84 190545.11 208534.3 206611.95 325605.44 323139.28 359118.97 374372.66 396915.34 267576.84 384776.12 359057.38 432589.53 342248.91 419019.47 410833.47 367840.22 402630.09 367351.16 319790.97 N-ACETYL-L-ASPARTIC ACID 5110.1 4589.26 3517.8 - 3764.54 46495.58 16362.37 15478.82 24259.91 - 6558.8 10655.89 7526.47 5605.69 - 85398.35 97948.73 98853.51 174206.17 159901.56 197749.7 - - - - - 10819.21 - - - 3469.08 - - - 5273.05 - 5749.66 D-Glucono-1-5-lactone - - - - - 20648.9 22559.76 18096.56 23677.26 22488.88 25364.82 31531.74 21414.21 30428.28 29071.79 17490.29 15615.14 19837.64 15548.11 20724.13 11070.87 17163.64 19440.32 21695.1 17551.29 8451.03 24927.88 28912.7 31313.24 24769.1 34573.23 19130.63 36592.55 39743.73 25934.31 35077.39 22569.24 ALPHA-D-GLUCOSE - 1933.92 - - 1735.56 10349601 13612131 12783976 15188419 13236288 16036875 18604634 19028898 21679874 20869592 16209597 15258132 15159179 9211338 10893488 10239907 12456599 11192485 15794752 13965488 15497208 16218352 17058792 19182670 20568738 17738600 20077534 22175502 19670502 15280016 13944167 14190363 L-Tyrosine - - 3665.55 - - 695551.25 595423.44 694248.5 714122.81 845073.19 825113.75 975179.06 1029464 1390664 1368573.38 1303688.25 1201992.12 1030590.69 637179.06 706227.19 682411.69 611622.44 568244.88 633757.19 732353.5 855718.44 869864.25 1231342.88 1024707.19 1003987.69 1066765.88 1044744.31 974649.19 1013821.69 773290.31 703764.31 726129.75 O-Phospho-L-serine - - - - - 27821.19 35889.78 20870.69 15698.53 12404.66 17958.59 19522.15 18376.12 27605.63 19563.23 50901.29 48735.73 45859.63 33260.41 40221.35 40355.99 16871.01 13394.25 9838.24 16616.81 20643.8 11625.25 17630.64 11612.83 13095.15 11974.62 6347.02 5463.72 14578.23 17712.24 23395.95 15202.89 citrate 49358.65 45673.14 80350.16 55952.56 47513.83 56145.2 45575.93 67880.01 39623.57 70259.88 53802.95 75142.93 54032.04 48181.32 54844.17 61397.78 69019.29 66703.8 59134.64 79445 92671.48 70997.75 44674.29 72992.62 50037.54 72461.71 57079.08 63652.67 66846.04 64910.65 51807.25 63864.44 66192.84 77720.04 55658.97 90843.2 78696.92 DEOXYCYTIDINE - - - - 3257.36 11410.38 10074.96 15884.27 11169.97 25564.61 16558.79 19719.1 27202.61 27278.06 26374.87 41340.43 21263.19 - - - - 12966.23 13875.99 12214.86 16783.04 15434.94 16871.85 30493.58 17204.36 22202.93 23210.86 - - 3788.9 1787.77 - - deoxyuridine 1480.29 - - - - 1884190.62 1810675.88 2141823.75 2040774.38 2286515.75 2160404.25 2272751.75 2193513 2818057 2631911 1668453 1420365 1594501.88 1673291.88 1533058.5 1926762.62 2301301.75 2270936.75 2061378 1726835.62 1526904.88 1416457.88 1292642.25 1125106.38 808345.69 736126.69 648429.94 753916.31 596630.12 2792568 2668845.75 2781867 Cystine - - - - - 87606.66 87359.57 96976.23 96630.65 113112.72 124580.32 162435.11 116875.21 140273.98 191362.83 52296.25 34932.07 47853.09 98041.8 86392.07 109891.41 134472.48 103330.98 144953.78 115764.41 112191.56 144840.77 142842.77 163883.94 150971.19 134175.2 155008.77 136900.28 164882.42 150323.02 144961.61 120719.3 thymidine 2099.23 1402.95 - - - 17692.1 31555.21 27591.18 25143.54 35464.19 34737.07 49843.46 42311.27 36777.55 42841.08 35096.76 35842.25 28790.62 27798.8 39519.33 31028.43 22799.96 27294.02 27119.82 14644.48 33440.7 41491.48 41356.04 47035.8 39929.65 53738.59 45567.09 69687.25 52523.11 20620.73 39523.79 42954.32 aminoimidazole carboxamide ribonucleotide - 1281.62 - - - 150325.12 175442.17 197047.81 202017.17 209112.25 206660.45 213083.52 267491.09 299594.88 314756.5 192979.69 186068.31 214110.05 212983.67 177280.17 185291.69 172661.3 172911.83 182497.86 206883.45 196894.2 226672 289777.84 280078.62 323609.22 243526.12 260285 356325.75 306157.34 218484 194619.44 180596.19 Sedoheptulose bisphosphate - 1618.87 - - 1628.13 204503.38 181436.52 179948.23 166163.23 149707.27 191334.05 160387.28 172255.55 141239.53 174896.14 142470.7 99946.9 133334.92 - - - 166259.33 150327.38 168523.61 177953.14 138601 155166.33 139415.02 135615.92 152619.38 147690.27 - - - - - - folate - - - - - 49305.08 22741.35 20851.16 35534.73 56980.86 36711.23 54818.01 40864.21 58472.7 69159.59 47748.48 50329.42 45928.58 37361.75 34370.07 44450.89 40099.86 36853.76 51748 46575.79 37480.98 51061.41 46740.22 58256.88 57959.86 43316.42 42572.25 27801.05 47728.3 33395.26 35552.9 24466.04 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pyruvate L-Alanine lactate Acetoacetate L-Serine MALEAMATE L-PROLINE 3-Methyl-2-oxobutanoic acid Fumarate L-Valine Succinate L-Threonine 4-HYDROXYBENZALDEHYDE Thymine Pyroglutamic acid 5-Oxoproline Citraconic acid L-Leucine L-Asparagine L-ORNITHINE Aspartate malate Hypoxanthine a-ketoglutarate L-Glutamine L-LYSINE L-Glutamate CITRAMALATE L-METHIONINE D-Ribose L-HISTIDINE 3-HYDROXY-3-METHYLGLUTARATE L-Phenylalanine arginine N-ACETYL-L-ASPARTIC ACID D-Glucono-1-5-lactone ALPHA-D-GLUCOSE L-Tyrosine O-Phospho-L-serine citrate DEOXYCYTIDINE deoxyuridine Cystine thymidine aminoimidazole carboxamide ribonucleotide Sedoheptulose bisphosphate folate METABOLITES_END #END