#METABOLOMICS WORKBENCH boryana_ptrva_20210422_055451 DATATRACK_ID:2588 STUDY_ID:ST001766 ANALYSIS_ID:AN002873 VERSION 1 CREATED_ON 03-03-2022 #PROJECT PR:PROJECT_TITLE Redox metabolism measurement in mammalian cells and tissues by quantitative PR:PROJECT_TITLE LC/MS method (part I) PR:PROJECT_SUMMARY This study aimed to optimize the detection of several key redox-reactive PR:PROJECT_SUMMARY metabolites from mammalian cells and tissues. We explored three different PR:PROJECT_SUMMARY chromatographic methods and optimized sample preparation, extraction buffer and PR:PROJECT_SUMMARY conditions as well as mass spectrometry detection parameters. The established PR:PROJECT_SUMMARY method was tested and validated using biologically relevant ROS-inducing PR:PROJECT_SUMMARY conditions. This study can be a valuable resource for the metabolomics PR:PROJECT_SUMMARY community. PR:INSTITUTE Boston Childrens Hospital PR:DEPARTMENT Pathology PR:LABORATORY Naama Kanarek PR:LAST_NAME Petrova PR:FIRST_NAME Boryana PR:ADDRESS 300 Longwood Av, Boston, MA, 2115, USA PR:EMAIL boryana.petrova@childrens.harvard.edu PR:PHONE 6173557433 PR:DOI http://dx.doi.org/10.21228/M81D6M #STUDY ST:STUDY_TITLE Application of the redox metabolite detection method for mammalian tissues (part ST:STUDY_TITLE I) ST:STUDY_SUMMARY This study was aimed at optimizing redox metabolites detection from mammalian ST:STUDY_SUMMARY tissues. Three different chromatographic conditions were compared as well as ST:STUDY_SUMMARY three different extraction conditions. This study was run on LUNA NH2 HILIC ST:STUDY_SUMMARY chromatography ST:INSTITUTE Boston Children's Hospital, Harvard Medical School ST:DEPARTMENT Pathology ST:LABORATORY Naama Kanarek ST:LAST_NAME Petrova ST:FIRST_NAME Boryana ST:ADDRESS 300 Longwood Av, Boston, MA, 2115, USA ST:EMAIL boryana.petrova@childrens.harvard.edu ST:PHONE 6173557433 ST:SUBMIT_DATE 2021-04-22 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - blank1 Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_blank_end1; Other=- SUBJECT_SAMPLE_FACTORS - blank2 Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_blank_end2; Other=- SUBJECT_SAMPLE_FACTORS - blank3 Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_blank_start1; Other=- SUBJECT_SAMPLE_FACTORS - blank4 Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_blank_start2; Other=- SUBJECT_SAMPLE_FACTORS - pooled sample QC 1/10 dilution Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP421_pool01x; Other=- SUBJECT_SAMPLE_FACTORS - pooled sample QC 1/10 dilution a Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP421_pool01x_a; Other=- SUBJECT_SAMPLE_FACTORS - pooled sample QC 1/3 dilution Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP420_pool03x; Other=- SUBJECT_SAMPLE_FACTORS - pooled sample Qca Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP419_pool1x_a; Other=- SUBJECT_SAMPLE_FACTORS - pooled sample QCb Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP419_pool1x_b; Other=- SUBJECT_SAMPLE_FACTORS - pooled sample QCc Factor:- RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP419_pool1x_c; Other=- SUBJECT_SAMPLE_FACTORS - kidney1 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP416; Other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - kidney2 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP417; Other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - kidney3 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP418; Other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - liver chunk1 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP413; Other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - liver chunk2 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP414; Other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - liver chunk3 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF2_LUNA_liver_kidney_BP415; Other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" #COLLECTION CO:COLLECTION_SUMMARY All animal care and experimental procedures were approved by the Institutional CO:COLLECTION_SUMMARY Animal Care and Use Committees of Boston Children’s Hospital. Mouse strain CO:COLLECTION_SUMMARY used was C57BL/6. Pure CSF samples were collected from the cisterna magna [39]. CO:COLLECTION_SUMMARY Blood samples were collected from the retromandibular vein. The samples were CO:COLLECTION_SUMMARY coagulated and centrifuged. Liver and kidney were collected and flash frozen. CO:COLLECTION_SUMMARY Tissue chunks were cut on a glass plate while kept chilled on top of dry ice. CO:COLLECTION_SUMMARY K562 cells used in this manuscript were authenticated by short tandem repeat CO:COLLECTION_SUMMARY analysis and tested negative for mycoplasma. Cells were cultured in RPMI CO:COLLECTION_SUMMARY (Genesee Scientific) up to a density of 2 Million cells per ml. For redox CO:COLLECTION_SUMMARY chemical treatment experiments, cells were seeded at 1 Million cells per ml cell CO:COLLECTION_SUMMARY density in 6-well plates and drugs were added for 4h at the following CO:COLLECTION_SUMMARY concentrations: methotrexate: 5 µM; oligomycin: 80 µg/ml; H2O2: 1 mM; diamide: CO:COLLECTION_SUMMARY 0.5 mM; DMSO, which served as control: 0.6 µl per 1 mL of cell culture media CO:COLLECTION_SUMMARY (equivalent to volume used for oligomycin). CO:SAMPLE_TYPE Liver/Kidney #TREATMENT TR:TREATMENT_SUMMARY Buffer Only #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were quenched as quickly as possible while working with the samples SP:SAMPLEPREP_SUMMARY at low temperatures. Cells were handled at 4ºC or on dry ice, extraction buffer SP:SAMPLEPREP_SUMMARY was pre-chilled at -20ºC. Samples were analyzed by LC-MS on the same day of SP:SAMPLEPREP_SUMMARY extraction (if impractical, best alternative is to store dried samples at SP:SAMPLEPREP_SUMMARY -80ºC). Unless indicated otherwise, 1 million cells or about 2 mg of tissue was SP:SAMPLEPREP_SUMMARY extracted per condition and a minimum of three replicates per condition was used SP:SAMPLEPREP_SUMMARY in each experiment. K562 cells that are non-adherent, were collected by brief SP:SAMPLEPREP_SUMMARY centrifugation at 4ºC using a table-top centrifuge (4,500 rpm, 1.5 min) and SP:SAMPLEPREP_SUMMARY washed briefly in 0.9% NaCl (high grade salt and LC-MS-grade water Fisher SP:SAMPLEPREP_SUMMARY Scientific W6500 or Sigma Aldrich 1.15333). 300 µl of prechilled extraction SP:SAMPLEPREP_SUMMARY buffer were added per sample. For tissues – chunks were crushed using a SP:SAMPLEPREP_SUMMARY hand-held homogenizer (VWR 47747-370) with several pulses while keeping the SP:SAMPLEPREP_SUMMARY samples on ice. 300 µl of prechilled extraction buffer was used per 2 mg of SP:SAMPLEPREP_SUMMARY tissue. Extraction buffer “A”: 40:40:20 of acetonitrile:methanol:water, SP:SAMPLEPREP_SUMMARY supplemented with 0.1M formic acid and isotopically-labeled internal standards SP:SAMPLEPREP_SUMMARY (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, SP:SAMPLEPREP_SUMMARY MSK-A2-1.2 and CNLM-6245-10). Extraction buffer “B”: 80% LC-MS-grade SP:SAMPLEPREP_SUMMARY methanol, 20% 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS SP:SAMPLEPREP_SUMMARY water and supplemented with isotopically labeled internal standards (17 amino SP:SAMPLEPREP_SUMMARY acids and isotopically labelled reduced glutathione, Cambridge Isotope SP:SAMPLEPREP_SUMMARY Laboratories, MSK-A2-1.2 and CNLM-6245-10). Extraction buffer “C” and “C + SP:SAMPLEPREP_SUMMARY Ellman’s”: Solution 1: 100% LC-MS Methanol Solution 2: 25mM Ammonium Acetate SP:SAMPLEPREP_SUMMARY and 2.5mM Na-Ascorbate in LC-MS water supplemented with isotopically labelled SP:SAMPLEPREP_SUMMARY reduced glutathione and isotopically labeled internal standards (17 amino acids SP:SAMPLEPREP_SUMMARY and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and SP:SAMPLEPREP_SUMMARY CNLM-6245-10). Ellman’s reagent (5,5′-Dithiobis(2-nitrobenzoic acid),D8130, SP:SAMPLEPREP_SUMMARY Sigma Aldrich): 20 mM in “Solution 2”. Final composition is 80% solution 1 SP:SAMPLEPREP_SUMMARY and 20% solution 2. Samples were vortexed briefly (10 sec) and sonicated for 3 SP:SAMPLEPREP_SUMMARY min in a 4ºC water bath. Samples were then centrifuged for 10 min, 4ºC, at SP:SAMPLEPREP_SUMMARY maximum speed on a benchtop centrifuge (Eppendorf) and the cleared supernatant SP:SAMPLEPREP_SUMMARY was transferred to a new tube. Samples were dried using a nitrogen dryer while SP:SAMPLEPREP_SUMMARY on ice, and special attention was given to minimize the time of drying and to SP:SAMPLEPREP_SUMMARY not let samples idle in the dryer (Reacti-Vap™ Evaporator, Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific, TS-18826) once the drying process was completed. Needles were SP:SAMPLEPREP_SUMMARY continuously adjusted to the surface of the liquid as the samples evaporated to SP:SAMPLEPREP_SUMMARY expedite the drying process. Samples were reconstituted in 30 µl LC-MS-grade SP:SAMPLEPREP_SUMMARY water by brief sonication in a 4ºC water bath. Extracted metabolites were spun SP:SAMPLEPREP_SUMMARY for 2 min at maximum speed on a bench-top centrifuge and cleared supernatant was SP:SAMPLEPREP_SUMMARY transferred to LC-MS micro vials (National Scientific, C5000-45B). A small SP:SAMPLEPREP_SUMMARY amount of each sample was pooled and serially diluted 3- and 10-fold to be used SP:SAMPLEPREP_SUMMARY as quality controls throughout the run of each batch. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY LUNA-NH2 chromatography: One ml of reconstituted sample was injected into a CH:CHROMATOGRAPHY_SUMMARY Luna® 3 µm NH2 100 Å, LC Column (150x2 mm, 3 µm particle size; Phenomenex, CH:CHROMATOGRAPHY_SUMMARY 00F-4377-B0) operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher CH:CHROMATOGRAPHY_SUMMARY Scientific, San Jose, CA). Chromatographic separation was achieved using the CH:CHROMATOGRAPHY_SUMMARY following conditions: buffer A was acetonitrile; buffer B was 5 mM ammonium CH:CHROMATOGRAPHY_SUMMARY acetate and 0.2% ammonium hydroxide. Gradient conditions were: 20 min linear CH:CHROMATOGRAPHY_SUMMARY gradient from 10% to 90% B; 20-25 min hold at 90% B; 25–26 min from 90% to 10% CH:CHROMATOGRAPHY_SUMMARY B; 26–34 min hold at 10% B. The column oven and autosampler tray were held at CH:CHROMATOGRAPHY_SUMMARY 30 °C and 4 °C, respectively CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Luna NH2 (150 x 2mm, 3um) CH:CHROMATOGRAPHY_TYPE Normal phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS HESI MS:ION_MODE UNSPECIFIED #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ppm MS_METABOLITE_DATA_START Samples blank1 blank2 blank3 blank4 pooled sample QC 1/10 dilution pooled sample QC 1/10 dilution a pooled sample QC 1/3 dilution pooled sample Qca pooled sample QCb pooled sample QCc kidney1 kidney2 kidney3 liver chunk1 liver chunk2 liver chunk3 Factors Factor:- Factor:- Factor:- Factor:- Factor:- Factor:- Factor:- Factor:- Factor:- Factor:- Factor:buffer C_Ell Factor:buffer C_Ell Factor:buffer C_Ell Factor:buffer C_Ell Factor:buffer C_Ell Factor:buffer C_Ell 2-aminoadipate 2805903.0000 1590946.0000 1872951.0000 11681165.0000 33371430.0000 38538330.0000 37545464.0000 14570521.0000 36102537.0000 12476173.0000 35584152.0000 106395704.0000 12913800.0000 2-aminobutyric_acid 641965.0000 7183726.0000 16752604.0000 17761747.0000 16645556.0000 2777945.0000 3846689.0000 3338245.0000 35076847.0000 39335573.0000 34503587.0000 2-hydroxybutyric_acid 57871328.0000 1836503.0000 3611071.0000 3691608.0000 2822793.0000 6738690.0000 7765867.0000 9057469.0000 2-hydroxyglutarate 2777257.0000 2300793.0000 67957789.0000 14683256.0000 3401374.0000 6449612.0000 10362184.0000 26731834.0000 19662521.0000 17833311.0000 27779006.0000 27549222.0000 19247237.0000 12984597.0000 22177255.0000 15116884.0000 2-oxoadipate 224837.0000 253817.0000 891954.0000 2636616.0000 2014659.0000 1846277.0000 1514360.0000 2109547.0000 2119263.0000 2176236.0000 2665155.0000 2286581.0000 2-oxobutyric_acid 3391439.0000 9920427.0000 10679487.0000 8781778.0000 7398220.0000 6530388.0000 4806002.0000 14731488.0000 13360180.0000 13119704.0000 3-hydroxybutyric_acid 2085986.0000 3198059.0000 3803044.0000 4037827.0000 2835280.0000 2358121.0000 2593783.0000 3355575.0000 5460520.0000 5231009.0000 3-hydroxyisobutyric_acid 3-methyl-2-oxobutyrate 3-methyl-2-oxopentanoic_acid 3-phosphoglycerate 2588148.0000 374868.0000 1268609.0000 2074935.0000 15383593.0000 7172998.0000 4882190.0000 8485852.0000 12132027.0000 5011460.0000 6286414.0000 9298965.0000 5849988.0000 3-ureidopropionate 6-phosphogluconate 195686.0000 127867.0000 520832.0000 3537000.0000 1886434.0000 1498149.0000 489491.0000 694560.0000 584799.0000 3633649.0000 5493826.0000 2570980.0000 acetoacetate acetylalanine 1301816.0000 5543055.0000 5631212.0000 5201164.0000 8034639.0000 9131121.0000 8580786.0000 3473678.0000 5356558.0000 2994197.0000 acetylaspartate acetylcarnitine 315258.0000 90787093.0000 84538408.0000 325972437.0000 880424849.0000 962035869.0000 991694362.0000 935027286.0000 978349643.0000 1170263917.0000 980917586.0000 1373680736.0000 763937469.0000 Acetyl CoA acetylglutamate acetylglutamine acetylglycine acetylmethionine acetylserine adenine 16454126.0000 11351628.0000 68422192.0000 139913535.0000 186951031.0000 198519112.0000 268451463.0000 242766818.0000 221428983.0000 114187018.0000 202758104.0000 98230877.0000 adenosine 89414957.0000 69992051.0000 315811403.0000 667990363.0000 880853210.0000 909831567.0000 1248398100.0000 1165028898.0000 1028627601.0000 534615610.0000 932005194.0000 473836439.0000 ADP 158479.0000 14750461.0000 275481.0000 4564892.0000 5598510.0000 16978413.0000 55649314.0000 46119906.0000 44776677.0000 34148629.0000 33680958.0000 33863776.0000 64024338.0000 60825394.0000 76273347.0000 ADP-ribose AICAR 128694.0000 761158.0000 632466.0000 608865.0000 179609.0000 387260.0000 361549.0000 1378275.0000 923175.0000 ALA_114 alanine 4284450.0000 13213947.0000 14464127.0000 14582942.0000 4277119.0000 4374292.0000 5350247.0000 30476528.0000 33621270.0000 20234138.0000 alanine-13C3-15N allantoin 2150972.0000 2048166.0000 9529057.0000 26495953.0000 27389921.0000 26797630.0000 22452791.0000 16384740.0000 23444727.0000 36017999.0000 49081413.0000 26903111.0000 Alpha-Ketoglutarate 2334394.0000 amidoadipate 2695906.0000 1590946.0000 1872951.0000 11681165.0000 33371430.0000 38538330.0000 37545464.0000 14570521.0000 36102537.0000 12476173.0000 35584152.0000 106395704.0000 12913800.0000 AMP 182468.0000 71230516.0000 3170566.0000 32901459.0000 59874639.0000 122168772.0000 458086826.0000 351617018.0000 334787319.0000 332693925.0000 385596973.0000 358755492.0000 397069936.0000 463112195.0000 346809889.0000 anserine arginine 307059.0000 3032494.0000 7178197.0000 19313173.0000 25275875.0000 38728214.0000 32266600.0000 51708607.0000 2218165.0000 672558.0000 2246170.0000 "arginine-13C6_15N4" 585331.0000 1637786.0000 1893492.0000 1565964.0000 2405309.0000 351213.0000 228805.0000 324830.0000 argininosuccinate 2354307.0000 906278.0000 3959413.0000 5773689.0000 6849429.0000 7731295.0000 5703623.0000 5122920.0000 7012433.0000 4435052.0000 5447901.0000 ascorbate 11064376.0000 21389330.0000 50920241.0000 58454526.0000 39983504.0000 12050025.0000 39443196.0000 107792779.0000 126098684.0000 50276798.0000 asparagine 1267273.0000 1258704.0000 1658034.0000 1283598.0000 1065849.0000 1575110.0000 1986703.0000 2221078.0000 1213824.0000 aspartate 3631173.0000 3158120.0000 12043301.0000 11798104.0000 20321023.0000 38126126.0000 21251226.0000 57565988.0000 41418245.0000 38527969.0000 53907435.0000 77485686.0000 74603565.0000 16326400.0000 19581685.0000 12338679.0000 aspartate-13C6-15N4 asymmetric dimethylarginine (ADMA) 2664465.0000 3449770.0000 1974228.0000 2341599.0000 4435498.0000 4633481.0000 2696278.0000 ATP 606473.0000 460983.0000 3675247.0000 9035842.0000 9938442.0000 9709552.0000 6918932.0000 6625689.0000 7103242.0000 16248470.0000 13619970.0000 12629260.0000 beta-alanine 1502967.0000 724247.0000 1700775.0000 1799603.0000 4031399.0000 5683298.0000 4074552.0000 betaine 381159968.0000 374618472.0000 1233523915.0000 3044517246.0000 3273519021.0000 3215077782.0000 2971461032.0000 2146779819.0000 3411389316.0000 2505444594.0000 4796105895.0000 3429151873.0000 biliverdin biotin Butyrylcarnitine 15135100.0000 13236007.0000 63405476.0000 175886892.0000 189101698.0000 193123130.0000 153755306.0000 138589777.0000 203474922.0000 234901344.0000 307374139.0000 195997503.0000 C5 Carnitines 3514576.0000 3211392.0000 17391366.0000 47204791.0000 47427498.0000 48717552.0000 32117684.0000 24463928.0000 33834190.0000 75123671.0000 89661767.0000 64723231.0000 Carbamoyl Aspartate carnitine 120287876.0000 103406532.0000 471775459.0000 1408976789.0000 1698302354.0000 1763898730.0000 2136178870.0000 2055431487.0000 2728017154.0000 1122495276.0000 1439467030.0000 957151242.0000 carnosine 408679.0000 533208.0000 979710.0000 1456868.0000 CDP CDP-choline choline 46623045.0000 42782877.0000 149196948.0000 537848666.0000 556114748.0000 542528335.0000 859702133.0000 896555937.0000 868828791.0000 288311383.0000 332177897.0000 283344893.0000 cis-aconitate 1637800.0000 1169252.0000 15194387.0000 17096748.0000 43629777.0000 119404236.0000 100899317.0000 84955708.0000 135369293.0000 141000787.0000 112901056.0000 80806424.0000 75041717.0000 83307773.0000 citrate 252137575.0000 9451176.0000 13640043.0000 14574539.0000 29135852.0000 106342019.0000 117510605.0000 118682081.0000 129385083.0000 117572804.0000 246115829.0000 84117518.0000 56488446.0000 61659433.0000 citrulline 900885.0000 648934.0000 785614.0000 790053.0000 276476.0000 766863.0000 1802988.0000 2135707.0000 1350363.0000 CMP 1500960.0000 569410.0000 2772298.0000 2221990.0000 2508543.0000 1731073.0000 2402181.0000 2332464.0000 3336997.0000 4081793.0000 2296012.0000 Coenzyme A CoproP_III creatine 82940173.0000 59660144.0000 413820653.0000 1121901763.0000 1386691259.0000 1420603677.0000 1992717305.0000 1871598328.0000 1831239679.0000 818369878.0000 864940285.0000 501084145.0000 creatinine 9390085.0000 7660749.0000 24876900.0000 97980330.0000 106017697.0000 114649739.0000 137029289.0000 112058974.0000 229720668.0000 72017149.0000 73051233.0000 59069843.0000 CTP cystathionine 457648.0000 2601940.0000 2908570.0000 2655442.0000 4820582.0000 4847919.0000 4652344.0000 Cysteic_acid cysteine cysteine_13C3_15N1 cysteine-glycine cystine 2582238.0000 3729485.0000 4351578.0000 13397301.0000 14599131.0000 7857491.0000 cytidine 98160.0000 577572.0000 604801.0000 894264.0000 962656.0000 1057821.0000 334235.0000 343158.0000 149909.0000 cytosine 1189756.0000 3694394.0000 3087444.0000 3378806.0000 5788666.0000 4663813.0000 6083165.0000 2102383.0000 2301336.0000 1227347.0000 dATP dCTP dCytidine DeuteroP dihydroorotate dihydroxyacetone_phosphate dimethylglicine 641964.0000 530383.0000 7184760.0000 17060215.0000 18040454.0000 16645531.0000 2920111.0000 4166297.0000 3338263.0000 34412590.0000 38713798.0000 35452034.0000 dTMP dTTP 482440.0000 2551955.0000 2363785.0000 4139336.0000 3160986.0000 3120197.0000 2648176.0000 2886923.0000 3389336.0000 3276989.0000 3370755.0000 dUMP erythrose_4-phosphate F6P_G1P 2453194.0000 5567402.0000 9602503.0000 34161178.0000 23845527.0000 22865038.0000 9226258.0000 9984334.0000 9378401.0000 36501656.0000 54938061.0000 35668882.0000 FAD folic_acid formyl_methionine fructose 13276249.0000 "Fructose 1_6-bisphosphate" fumarate 2720723.0000 2569429.0000 14238416.0000 5375487.0000 5775571.0000 8190047.0000 19278319.0000 44977617.0000 40216976.0000 37521718.0000 38882813.0000 34205010.0000 29224951.0000 62771770.0000 71813058.0000 34442139.0000 gamma-aminobutyric_acid 173201.0000 1770870.0000 1627693.0000 1413616.0000 2139246.0000 1610614.0000 2495714.0000 1925996.0000 1989449.0000 1119578.0000 gamma-aminolevulinic_acid gamma-glutamyl-alanine 277463.0000 2711196.0000 5551223.0000 6563750.0000 6778862.0000 11997959.0000 3480180.0000 2630773.0000 11112857.0000 5594341.0000 11669986.0000 GAR GDP 320569.0000 1318404.0000 1490697.0000 1415915.0000 1555016.0000 1492075.0000 1733148.0000 1291745.0000 1031251.0000 1772341.0000 glucose 8212123.0000 8726237.0000 47116243.0000 117170231.0000 106798704.0000 98688668.0000 5326509.0000 4647190.0000 4202721.0000 195581597.0000 271617240.0000 175916238.0000 Glucose 6-phosphate 2380602.0000 5567402.0000 9863233.0000 34161178.0000 23845527.0000 22865038.0000 9536075.0000 10252707.0000 9343792.0000 35612257.0000 55091318.0000 34067469.0000 glutamate 358007.0000 410338.0000 7442900.0000 1620876.0000 34719117.0000 54977599.0000 47622732.0000 106293062.0000 90745441.0000 87017414.0000 135849665.0000 149846331.0000 139681671.0000 47957110.0000 48989477.0000 34063597.0000 "glutamate-13C5_15N" glutamine 502344.0000 6002551.0000 6189857.0000 33451220.0000 89556827.0000 90807510.0000 83260600.0000 27560765.0000 23227834.0000 33028486.0000 168687389.0000 173276287.0000 132223987.0000 glyceraldehyde_3-phosphate 202198.0000 glycerate 66186616.0000 68966189.0000 178358836.0000 376655194.0000 330628983.0000 338576534.0000 378746745.0000 388199902.0000 363246515.0000 322007436.0000 286144279.0000 346650516.0000 Glycerol 3-Phosphate 947969.0000 2618100.0000 3481753.0000 3026090.0000 142448.0000 678764.0000 887941.0000 5553639.0000 7970716.0000 4763610.0000 Glycerophosphocholine 153371789.0000 154371617.0000 392515286.0000 1664029051.0000 2325458148.0000 2455222786.0000 3493686817.0000 2579225488.0000 5940546072.0000 359378509.0000 512109363.0000 253148278.0000 glycine 317518.0000 486853.0000 1238512.0000 1143795.0000 1494446.0000 glycine_13C2-15N GMP 236999.0000 4714899.0000 9220236.0000 17805080.0000 80271670.0000 57723218.0000 50976850.0000 57215382.0000 62408026.0000 53468859.0000 65661553.0000 74473122.0000 62685602.0000 GSH GSH-13C2_15N GSH_13C2-15N-Ellmans GSH_Ellmans 1925075.0000 670349.0000 477600.0000 5687405.0000 11124492.0000 4333961.0000 GSSG 386649.0000 5302409.0000 15471992.0000 15517453.0000 13914649.0000 1772000.0000 10115406.0000 4897135.0000 26759925.0000 45411820.0000 17624978.0000 GTP guanidinoacetate guanine 235825.0000 1154315.0000 1134421.0000 1358069.0000 2198479.0000 2267710.0000 2451785.0000 guanosine histidine 827260.0000 502650.0000 7186871.0000 21384713.0000 20505542.0000 19034103.0000 7647584.0000 7020881.0000 7515144.0000 40580181.0000 44962438.0000 32506915.0000 histidine-13C6-15N3 homocitrulline homocysteine hydroxyproline 286168.0000 1301816.0000 5543055.0000 5631212.0000 5201164.0000 8034639.0000 9131121.0000 8580786.0000 3473678.0000 5356558.0000 2994197.0000 hypotaurine 251128.0000 1244452.0000 1184129.0000 1400940.0000 225413.0000 1766456.0000 1214723.0000 311470.0000 2551349.0000 1286448.0000 hypoxanthine 17988846.0000 13621958.0000 88610491.0000 215030056.0000 322134274.0000 365217110.0000 420350596.0000 420321184.0000 555067743.0000 108891026.0000 218251566.0000 99514934.0000 IMP 8105269.0000 14415853.0000 24335503.0000 120354272.0000 88750457.0000 79401451.0000 100342601.0000 137655475.0000 126120816.0000 51395746.0000 76454697.0000 69914584.0000 indolelactate inosine 248024.0000 1292998.0000 5457719.0000 5539283.0000 5398073.0000 6584226.0000 7862990.0000 6172624.0000 3016283.0000 8227929.0000 4041854.0000 isoleucine 4141212.0000 4237832.0000 18762626.0000 52456781.0000 47369010.0000 53061469.0000 40131541.0000 38268000.0000 39363859.0000 73366503.0000 82097893.0000 73997083.0000 Isoleucine-13C6-15N 385289.0000 730347.0000 1585634.0000 1678656.0000 2144951.0000 1860941.0000 2269500.0000 1556860.0000 1150380.0000 1337682.0000 Itaconic Acid 25059317.0000 Kynurenic Acid kynurenine lactatic_acid 3234969.0000 6028789.0000 33382706.0000 85550895.0000 76566082.0000 76038710.0000 2762785.0000 3696837.0000 5844006.0000 147867953.0000 208706117.0000 137228211.0000 leucine 7437338.0000 6086049.0000 26650731.0000 64914767.0000 76368030.0000 71993388.0000 50353173.0000 46978292.0000 58895183.0000 101220605.0000 117573695.0000 87507101.0000 leucine-13C6-15N 375227.0000 1141322.0000 1291418.0000 1290396.0000 1679826.0000 1543113.0000 1521973.0000 1159605.0000 895305.0000 1085492.0000 L-ureidosuccinic_acid L-Ureidosuccinic acid lysine 2159561.0000 1134149.0000 1019449.0000 7117167.0000 23204001.0000 21770528.0000 20548279.0000 12246098.0000 14996112.0000 11080151.0000 36655433.0000 36788469.0000 34237098.0000 lysine-13C6-15N2 malate 6654909.0000 5432089.0000 175674622.0000 39077637.0000 29936165.0000 39678563.0000 102483112.0000 257773757.0000 219593807.0000 200562334.0000 205201448.0000 192905138.0000 142007077.0000 331500671.0000 388958100.0000 186189853.0000 mannose 16480304.0000 29766049.0000 28261764.0000 64852704.0000 196752198.0000 234724467.0000 245362474.0000 427011998.0000 389933813.0000 515294844.0000 20905650.0000 21202011.0000 17598129.0000 methionine 1247051.0000 1014932.0000 4948183.0000 17512626.0000 16861043.0000 17612116.0000 21624233.0000 23034477.0000 22180280.0000 14844536.0000 13651479.0000 11008926.0000 methionine-13C5-15N 302693.0000 456627.0000 731138.0000 706712.0000 942023.0000 870892.0000 361081.0000 735404.0000 777614.0000 Methionine sulfoxide methylthioadenosine 1191807.0000 2729716.0000 3293097.0000 3375099.0000 2626050.0000 3110715.0000 2613738.0000 3536464.0000 6750002.0000 3462732.0000 myo-inositol N2-acetyllysine 1711088.0000 1228118.0000 1596369.0000 921511.0000 1325301.0000 2555242.0000 1299247.0000 N-acetylcysteine N-acetylglutamate N-acetylmethionine N-acetylornithine N-acetylphenylalanine NAD+ 8349210.0000 7809869.0000 27116188.0000 72088965.0000 77872835.0000 80554661.0000 79812911.0000 87324745.0000 99330313.0000 71383613.0000 80579261.0000 83633737.0000 NADH 5199525.0000 17738452.0000 16368839.0000 16769644.0000 6993054.0000 8197296.0000 10531240.0000 23307884.0000 20720211.0000 39231383.0000 NADP 2135063.0000 6142726.0000 7566291.0000 9150436.0000 5880415.0000 7167970.0000 8992688.0000 10464992.0000 7435538.0000 8064540.0000 NADPH niacinamide 363755874.0000 373944868.0000 1293439501.0000 2927935822.0000 2714177105.0000 2485491848.0000 2145394896.0000 2006547844.0000 1609418252.0000 3444438421.0000 5221887530.0000 1770085695.0000 O-phosphoethanolamine 2336667.0000 2633103.0000 6931629.0000 23490085.0000 27314212.0000 26412648.0000 43185413.0000 43390123.0000 51481855.0000 7536253.0000 7910418.0000 5999251.0000 ornithine 224562.0000 3437249.0000 3997756.0000 12659583.0000 19107564.0000 3712055.0000 1621372.0000 4974757.0000 21436835.0000 21600703.0000 12845420.0000 orotate palmitate p-aminobenzoate Pantothenate 1861227.0000 1676117.0000 2186609.0000 4292126.0000 2997235.0000 3439593.0000 373872.0000 275496.0000 PBG 1218581.0000 1334767.0000 2479433.0000 1363367.0000 2063285.0000 Phenylacetyl glutamine phenylalanine 120020.0000 3779745.0000 4189879.0000 15919839.0000 43298610.0000 44492359.0000 45362149.0000 36367226.0000 34817761.0000 39404283.0000 59209657.0000 69117423.0000 55672886.0000 phenylalanine-13C9-15N 710260.0000 1575218.0000 1558603.0000 2184945.0000 2338567.0000 2287031.0000 2780564.0000 1734411.0000 1884026.0000 1858328.0000 phosphocholine 516432.0000 24169107.0000 20505797.0000 105759796.0000 307885667.0000 509485054.0000 557872587.0000 673580101.0000 574104038.0000 942942104.0000 250219090.0000 476183467.0000 325314780.0000 phosphocreatine 521971.0000 804766.0000 1096517.0000 887481.0000 1514655.0000 956296.0000 1047504.0000 1553932.0000 1095099.0000 phosphoenolpyruvate phosphoethanolamine 503457.0000 1006189.0000 3735949.0000 15033295.0000 15897191.0000 16862703.0000 25894052.0000 26425498.0000 33135738.0000 4961216.0000 6421657.0000 3899383.0000 phosphoserine Picolinate Pipecolate proline 6013704.0000 6199564.0000 42638377.0000 143600729.0000 140097727.0000 127930871.0000 78207355.0000 77611464.0000 73786885.0000 274693260.0000 238171935.0000 184416221.0000 proline-13C5-15N 380945.0000 1646489.0000 2423594.0000 2238253.0000 2607913.0000 3130942.0000 2007952.0000 2145436.0000 2179256.0000 2292066.0000 propionylcarnitine 6399550.0000 5404809.0000 40969385.0000 108099438.0000 113966603.0000 113378592.0000 63691048.0000 53482336.0000 71805043.0000 181149897.0000 218421483.0000 168250665.0000 ProtoP_IX PRPP pseudouridine 745869.0000 755982.0000 2666636.0000 7852447.0000 8503228.0000 8725637.0000 11378459.0000 10113768.0000 14474262.0000 6536968.0000 10082349.0000 4773991.0000 Pyridoxal Pyridoxine pyruvate Riboflavin Ribose 1-phosphate 602521.0000 1634764.0000 4782809.0000 14371336.0000 12326762.0000 11782576.0000 7460070.0000 7810756.0000 5577753.0000 17846564.0000 27415928.0000 18487693.0000 Ribose_Ribulose 5-phosphate 653913.0000 1634764.0000 4782809.0000 14371336.0000 12326762.0000 11782576.0000 7460070.0000 7810756.0000 5577753.0000 17846564.0000 27415928.0000 18487693.0000 S-Adenosyl-L-Homocysteine 845098.0000 2703106.0000 3532402.0000 3209589.0000 796129.0000 526082.0000 1296327.0000 6429978.0000 9106146.0000 7466051.0000 S-Adenosyl-L-methionine 1764680.0000 6700055.0000 6909374.0000 7227593.0000 7258095.0000 7666240.0000 7803621.0000 7117454.0000 11113842.0000 4743011.0000 sarcosine Sedoheptulose-7-phosphate 664743.0000 2238183.0000 3578265.0000 14123689.0000 9831464.0000 8569847.0000 3981202.0000 5832116.0000 5210075.0000 15819780.0000 25334853.0000 10515808.0000 serine 99089.0000 706113.0000 633902.0000 890608.0000 832335.0000 733647.0000 941037.0000 917079.0000 985584.0000 227948.0000 serine-13C3-15N sorbitol 43790793.0000 36825429.0000 19462908.0000 174284999.0000 312886169.0000 365476732.0000 203369257.0000 13666111.0000 1385079536.0000 12520403.0000 33089316.0000 12843980.0000 succinate 48081035.0000 2756515.0000 8753152.0000 8903231.0000 36417188.0000 90227454.0000 81037559.0000 81194545.0000 68421956.0000 66515505.0000 70516384.0000 96153109.0000 134710684.0000 91677380.0000 taurine 718033750.0000 1032457054.0000 1451437596.0000 2749922715.0000 2391934376.0000 2334288043.0000 1933607200.0000 1780650768.0000 2005340203.0000 2705996611.0000 3427449234.0000 3484709344.0000 thiamine threonine 1141140.0000 4963889.0000 5025230.0000 4761621.0000 4550910.0000 4490409.0000 5717247.0000 5252915.0000 6498266.0000 4998234.0000 threonine-13C4-15N thymidine 277513.0000 2213840.0000 4794553.0000 7446710.0000 8150149.0000 16367101.0000 14013596.0000 11660572.0000 1354370.0000 2758147.0000 274278.0000 trans-urocanic acid 1067506.0000 708437.0000 27690315.0000 1026507.0000 698142.0000 2108669.0000 2178044.0000 3006891.0000 2490977.0000 1153904.0000 3106523.0000 2394144.0000 3463738.0000 1400183.0000 3238288.0000 trimethyllysine 598556.0000 1634764.0000 4782809.0000 14371336.0000 12326762.0000 11782576.0000 7460070.0000 7810756.0000 5577753.0000 17846564.0000 27415928.0000 18487693.0000 tryptophan 492885.0000 489941.0000 3614490.0000 9593957.0000 9244842.0000 8204101.0000 7492279.0000 7268655.0000 9575195.0000 11686349.0000 13450416.0000 11123382.0000 tyrosine 323181.0000 1959215.0000 9228279.0000 10017532.0000 9522471.0000 9542502.0000 8413361.0000 9121226.0000 12278779.0000 12987853.0000 9232054.0000 tyrosine-13C9-15N 184568.0000 UDP 1206409.0000 929745.0000 7217798.0000 21015033.0000 21913367.0000 23368610.0000 16155339.0000 17994432.0000 18152309.0000 29113209.0000 40665123.0000 20572741.0000 UDP-GlcNAc 2873332.0000 3058985.0000 3026224.0000 1537400.0000 1925956.0000 1245070.0000 4658975.0000 6297561.0000 3053145.0000 UDP-hexose 2880223.0000 2836247.0000 12735121.0000 35326078.0000 37201512.0000 35340147.0000 26192841.0000 28873685.0000 28340913.0000 47607913.0000 69778704.0000 35646505.0000 UMP 4680498.0000 329294.0000 5764796.0000 13795612.0000 24214969.0000 125986956.0000 69566739.0000 55527188.0000 29393941.0000 40802851.0000 26915656.0000 100900797.0000 150247533.0000 112921752.0000 uracil 12487887.0000 10691212.0000 37581104.0000 168385259.0000 196074225.0000 210188471.0000 348758516.0000 362251913.0000 422834799.0000 7291992.0000 12559256.0000 9815233.0000 uric acid 10552739.0000 15144426.0000 17056225.0000 29364781.0000 21316734.0000 17194901.0000 8065388.0000 8793327.0000 7773124.0000 28313463.0000 48878636.0000 35018424.0000 uridine 20788294.0000 20814796.0000 86467209.0000 222003682.0000 231599891.0000 229664149.0000 199189606.0000 218246356.0000 219118545.0000 244070580.0000 423318655.0000 207702241.0000 UTP 202782.0000 204232.0000 346074.0000 517178.0000 580691.0000 1041845.0000 1642535.0000 1208559.0000 valine 5959844.0000 10427530.0000 20707085.0000 12189714.0000 12533872.0000 12736024.0000 11463990.0000 8527326.0000 23900244.0000 14241936.0000 19422770.0000 valine-13C5-15N 166602.0000 189164.0000 xanthine 12734443.0000 12028409.0000 18643445.0000 27812867.0000 25883602.0000 24991739.0000 21402928.0000 22762720.0000 24850994.0000 22702097.0000 46455515.0000 30532097.0000 xanthosine 4399664.0000 6110801.0000 9247750.0000 17991142.0000 10675299.0000 10026136.0000 2791371.0000 1290747.0000 1071000.0000 17259048.0000 29053086.0000 24667616.0000 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 2-aminoadipate 2-aminobutyric_acid 2-hydroxybutyric_acid 2-hydroxyglutarate 2-oxoadipate 2-oxobutyric_acid 3-hydroxybutyric_acid 3-hydroxyisobutyric_acid 3-methyl-2-oxobutyrate 3-methyl-2-oxopentanoic_acid 3-phosphoglycerate 3-ureidopropionate 6-phosphogluconate acetoacetate acetylalanine acetylaspartate acetylcarnitine Acetyl CoA acetylglutamate acetylglutamine acetylglycine acetylmethionine acetylserine adenine adenosine ADP ADP-ribose AICAR ALA_114 alanine alanine-13C3-15N allantoin Alpha-Ketoglutarate amidoadipate AMP anserine arginine "arginine-13C6,15N4" argininosuccinate ascorbate asparagine aspartate aspartate-13C6-15N4 asymmetric dimethylarginine (ADMA) ATP beta-alanine betaine biliverdin biotin Butyrylcarnitine C5 Carnitines Carbamoyl Aspartate carnitine carnosine CDP CDP-choline choline cis-aconitate citrate citrulline CMP Coenzyme A CoproP_III creatine creatinine CTP cystathionine Cysteic_acid cysteine cysteine_13C3_15N1 cysteine-glycine cystine cytidine cytosine dATP dCTP dCytidine DeuteroP dihydroorotate dihydroxyacetone_phosphate dimethylglicine dTMP dTTP dUMP erythrose_4-phosphate F6P_G1P FAD folic_acid formyl_methionine fructose "Fructose 1,6-bisphosphate" fumarate gamma-aminobutyric_acid gamma-aminolevulinic_acid gamma-glutamyl-alanine GAR GDP glucose Glucose 6-phosphate glutamate "glutamate-13C5,15N" glutamine glyceraldehyde_3-phosphate glycerate Glycerol 3-Phosphate Glycerophosphocholine glycine glycine_13C2-15N GMP GSH GSH-13C2_15N GSH_13C2-15N-Ellmans GSH_Ellmans GSSG GTP guanidinoacetate guanine guanosine histidine histidine-13C6-15N3 homocitrulline homocysteine hydroxyproline hypotaurine hypoxanthine IMP indolelactate inosine isoleucine Isoleucine-13C6-15N Itaconic Acid Kynurenic Acid kynurenine lactatic_acid leucine leucine-13C6-15N L-ureidosuccinic_acid L-Ureidosuccinic acid lysine lysine-13C6-15N2 malate mannose methionine methionine-13C5-15N Methionine sulfoxide methylthioadenosine myo-inositol N2-acetyllysine N-acetylcysteine N-acetylglutamate N-acetylmethionine N-acetylornithine N-acetylphenylalanine NAD+ NADH NADP NADPH niacinamide O-phosphoethanolamine ornithine orotate palmitate p-aminobenzoate Pantothenate PBG Phenylacetyl glutamine phenylalanine phenylalanine-13C9-15N phosphocholine phosphocreatine phosphoenolpyruvate phosphoethanolamine phosphoserine Picolinate Pipecolate proline proline-13C5-15N propionylcarnitine ProtoP_IX PRPP pseudouridine Pyridoxal Pyridoxine pyruvate Riboflavin Ribose 1-phosphate Ribose_Ribulose 5-phosphate S-Adenosyl-L-Homocysteine S-Adenosyl-L-methionine sarcosine Sedoheptulose-7-phosphate serine serine-13C3-15N sorbitol succinate taurine thiamine threonine threonine-13C4-15N thymidine trans-urocanic acid trimethyllysine tryptophan tyrosine tyrosine-13C9-15N UDP UDP-GlcNAc UDP-hexose UMP uracil uric acid uridine UTP valine valine-13C5-15N xanthine xanthosine METABOLITES_END #END