#METABOLOMICS WORKBENCH boryana_ptrva_20210422_082036_mwtab.txt DATATRACK_ID:2591 STUDY_ID:ST001768 ANALYSIS_ID:AN002875 PROJECT_ID:000000 VERSION 1 CREATED_ON May 4, 2021, 4:08 pm #PROJECT 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:FIRST_NAME Boryana PR:ADDRESS 300 Longwood Av, Boston, MA, 2115, USA PR:EMAIL boryana.petrova@childrens.harvard.edu PR:PHONE 6173557433 PR:PROJECT_TITLE Redox metabolism measurement in mammalian cells and tissues by quantitative PR:PROJECT_TITLE LC/MS method (part X) PR:LAST_NAME Petrova #STUDY ST:STUDY_TITLE Application of the redox metabolite detection method for mammalian tissues (part ST:STUDY_TITLE III) 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 ZIC-pHILIC 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 #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]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - blank1 Factor:- RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP_blank_start1_20201010100637; other=- SUBJECT_SAMPLE_FACTORS - blank2 Factor:- RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP_blank_start2_20201010103707; other=- SUBJECT_SAMPLE_FACTORS - liver chunk1 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP419; other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - liver chunk2 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP420; other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - liver chunk3 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP421; other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - kidney1 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP422; other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - kidney2 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP423; other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - kidney3 Factor:buffer C_Ell RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP424; other="isotopically labelled amino acids, GSH-13C2-15N, aminopterin" SUBJECT_SAMPLE_FACTORS - pooled sample Qca Factor:- RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP431_pool1x_a; other=- SUBJECT_SAMPLE_FACTORS - pooled sample QCb Factor:- RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP431_pool1x_b; other=- SUBJECT_SAMPLE_FACTORS - pooled sample QCc Factor:- RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP431_pool1x_c; other=- SUBJECT_SAMPLE_FACTORS - pooled sample QC 1/3 dilution Factor:- RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP432_pool03; other=- SUBJECT_SAMPLE_FACTORS - pooled sample QC 1/10 dilution a Factor:- RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP433_pool01x; other=- SUBJECT_SAMPLE_FACTORS - pooled sample QC 1/10 dilution b Factor:- RAW_FILE_NAME=20201009_QEF1_HILIC_liver_kidney_BP433_pool01x_a; other=- #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 ZIC-pHILIC chromatography: One ml of reconstituted sample was injected into a CH:CHROMATOGRAPHY_SUMMARY ZIC-pHILIC 150 × 2.1 mm (5 µm particle size) column (EMD Millipore) CH:CHROMATOGRAPHY_SUMMARY operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San CH:CHROMATOGRAPHY_SUMMARY Jose, CA). Chromatographic separation was achieved using the following CH:CHROMATOGRAPHY_SUMMARY conditions: buffer A was acetonitrile; buffer B was 20 mM ammonium carbonate, CH:CHROMATOGRAPHY_SUMMARY 0.1% ammonium hydroxide. Gradient conditions were: linear gradient from 20% to CH:CHROMATOGRAPHY_SUMMARY 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B. The CH:CHROMATOGRAPHY_SUMMARY column oven and autosampler tray were held at 25 °C and 4 °C, CH:CHROMATOGRAPHY_SUMMARY respectively. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME EMD Millipore ZIC HILIC (150 x 2.1mm, 5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS HESI #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ppm MS_METABOLITE_DATA_START Samples blank1 blank2 liver chunk1 liver chunk2 liver chunk3 kidney1 kidney2 kidney3 pooled sample Qca pooled sample QCb pooled sample QCc pooled sample QC 1/3 dilution pooled sample QC 1/10 dilution a pooled sample QC 1/10 dilution b Factors 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 Factor:- Factor:- Factor:- Factor:- Factor:- Factor:- 1-methylnicotinamide N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F "2,3-BPG" N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F 2-aminobutyric_acid N/F N/F 10861597 8939178 14403029 N/F N/F N/F 11769683 7161832 11276489 4376367 N/F N/F 2-hydroxybutyric_acid N/F N/F 10532525 10293352 11125685 N/F N/F N/F 3290697 N/F 2163622 N/F N/F N/F 2-hydroxyglutarate N/F N/F 273556828 236389639 245753957 273999556 261677621 254567325 239362502 245309770 249864005 105633414 37736869 34463005 2-oxoadipate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F 2-oxobutyric_acid N/F N/F 2560846 2055699 2326907 2310297 1335703 2403109 2212093 1858333 1914925 879075 N/F N/F 3-hydroxybutyric_acid N/F N/F 12320919 13399938 12156463 5304788 4816157 6084483 8701032 10153001 8932398 2940385 695165 946471 3-hydroxyisobutyric_acid N/F N/F 2420234 2516257 1317214 1042265 1067542 1183864 1671019 861093 1605286 618927 122642 250406 3-methyl-2-oxopentanoic_acid N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F 3-phosphoglycerate 1109512 N/F 7638783 9536494 4230159 8153229 6430541 6024814 6895151 6710025 6373925 1103282 N/F N/F 3-ureidopropionate N/F N/F 10078857 11999401 14762064 9149241 8828538 9665333 11145202 10144902 11613286 2813665 838624 957272 5-Hydroxyindole-3-Acetate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F 6-phosphogluconate N/F N/F 470248 710730 333957 65749 44574 85346 233037 218331 260382 32054 N/F N/F acetoacetate N/F N/F 2725311 2517106 2738238 2851955 2284061 2601536 2344446 2299248 2700790 767575 324060 N/F Acetyl CoA N/F N/F 21387260 15084950 16969795 2466623 2643094 2526872 10672593 9836427 10662396 2787663 737414 775192 acetylalanine N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F acetylaspartate N/F N/F 282672 N/F 111673 201461 100010 252513 146836 123592 126465 127005 N/F N/F acetylcarnitine N/F N/F 2055368897 2321584898 1504491311 1415888618 1386782386 1593852534 1724695693 1685658906 1659221428 539548166 174525736 194580041 acetylglutamate N/F N/F 38815437 39497993 33323775 1211073 1088666 1246047 19171141 19611812 17629210 6479998 2084646 2005772 acetylglycine N/F N/F N/F N/F N/F N/F N/F N/F 2122361 1830328 N/F N/F N/F N/F acetylmethionine N/F N/F 285718 494453 200036 238942 151248 136292 281811 232102 242986 N/F N/F N/F acetylserine N/F N/F 4031302 4164188 3366152 1303150 1820431 1054578 2496799 2212608 2669367 836317 292929 290621 adenine N/F N/F 32627523 34294608 36074665 114057208 108093825 116657343 74005182 78251909 77769719 25644962 7749929 7574140 adenosine N/F N/F 734649858 1150673731 639605760 1286787373 1175370033 925379958 980396584 1000641452 1014335510 362095206 126375080 124358413 ADP-ribose N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F ADP N/F N/F 38098266 31074973 39659802 16131590 14822849 15518384 25574305 25202962 25614529 7533302 2346387 2295532 AICAR N/F N/F 370475 643652 529705 170604 180981 181200 331685 366327 304325 98622 29726 26601 alanine-13C3-15N N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F alanine N/F N/F 274939677 267529371 189765884 98713534 99863176 81518780 161717694 166394737 161370115 51395127 15569045 16115437 allantoin N/F N/F 4080386 5602740 2625483 1227836 992985 1380277 2357025 2363963 2279239 1989856 1785042 1484282 alpha-keto-isovalerate N/F N/F N/F N/F N/F N/F N/F N/F N/F 1384666 N/F N/F N/F N/F Alpha-Ketoglutarate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Alpha-Lipoic Acid N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F amidoadipate N/F N/F 117995316 354475215 39812331 31344553 79501885 26991144 113238282 109725569 116071931 33793619 5959197 6530274 AMP N/F N/F 485265499 602448565 325211126 131513934 132188370 144955447 262603758 268255692 272387520 64201708 17907048 19063953 anserine N/F N/F 7651351 6945979 5260105 58794542 63025702 57963033 31047739 32889466 34710011 5054294 582962 690578 arginine-13C6-15N4 158191 N/F 24261238 21519845 28599538 26672342 24269629 22976574 21148611 24478328 25196878 9527537 3427731 3008620 arginine 6646655 2265302 77434689 34403855 71609557 436297157 470495855 406051310 232532460 277895640 276681259 106571091 40264950 36630257 argininosuccinate N/F N/F 22728991 15401056 16138235 11924698 10218822 6724857 13004757 13653262 14564193 4194738 189342 199123 ascorbate N/F N/F 415792868 422385400 237976719 167374673 104798072 136355052 150090421 259785005 285529732 108974885 27378124 11569913 asparagine N/F N/F 8824630 7827988 6814025 6011544 5346597 5880404 6463152 6756077 6469330 2054499 528233 471266 aspartate-13C6-15N4 N/F N/F N/F 153146 239730 519745 277606 401382 153529 N/F N/F 223595 N/F N/F aspartate N/F N/F 25703735 26441385 18477156 135557531 158845806 166470024 65378708 63946003 66180489 49188347 14010317 14936377 asymmetric dimethylarginine (ADMA) N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F ATP N/F N/F 8592691 6093031 5782050 2790078 2554913 2445027 4623783 4528642 4558920 1249885 315698 347395 beta-alanine N/F N/F 31424078 32175989 22495292 4307627 4200913 4089573 16895696 17339116 14891384 5353114 1420894 1415673 betaine N/F N/F 8277100588 14766504096 10147762114 6932487617 4716198873 7959372274 8912605690 8891167765 8893715987 2990785046 935072818 1034089520 biotin N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Butyrylcarnitine N/F N/F 388893248 395167119 303018481 177369814 161740570 218345734 272736541 263824594 264609509 79094855 23034198 25944590 C5 Carnitines N/F N/F 117654003 104707215 95789362 35125314 24594154 31607050 65557318 64036407 68118847 18192928 4937871 5139927 cAMP N/F N/F 1813568 1433475 1374472 563413 419637 547384 978463 812093 975804 215532 11842 N/F carnitine 56513 N/F 2607797645 2791857932 2201893055 3390260418 3186768160 3930488323 3050322398 2940828663 2989233567 1002499219 333004970 362178363 carnosine N/F N/F 3707820 4239274 4353797 50478644 52685364 63389646 28322760 30612133 29090282 7893931 1054100 1012306 CDP-choline N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F CDP N/F N/F 107099 79994 75556 36064 25622 50430 73367 51071 59243 N/F N/F N/F choline N/F N/F 26575441 34614354 18263738 113447401 89266280 159678927 83727430 87623714 88151233 36411459 18220507 17282085 cis-aconitate N/F N/F 1477094 1545000 1734420 6492084 1956130 15325279 4100274 5623231 4644978 1362981 395510 437142 citrate N/F N/F 15222916 N/F N/F 18821422 12315541 24687477 24566137 14577343 19678631 3571916 N/F N/F citrulline N/F N/F 97376912 84999611 72085881 34454253 23712240 28598567 58613137 57431368 56930431 16265717 2871893 3036647 CMP N/F N/F 1812548 1819978 1169182 818741 975806 905824 1184912 1138535 1198662 390729 106747 104561 CoA-Ellmans N/F N/F 1214374 1315607 1497254 475055 621117 559529 893769 812968 533044 168591 N/F 111305 Coenzyme A N/F N/F 364813 465785 413139 N/F N/F N/F 240314 476622 451651 N/F N/F N/F creatine N/F N/F 1800523370 1749962024 1216787644 2805707624 2735751008 2202074504 2084566951 2059650398 2039708380 682692475 219813777 235888075 creatinine N/F N/F 50170333 44468100 37691562 67920868 51120281 98400814 60559832 57126978 55151294 17433193 5280214 5441053 CTP N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F cystathionine N/F N/F 23131016 21808350 17167583 1123522 1084158 717571 12731354 12564432 12491595 1701574 345090 373348 cysteic_acid N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F cysteine-glycine N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F cysteine_13C3_15N1 N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F cysteine N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F cystine N/F N/F N/F N/F N/F 13811411 2656234 1271142 2915387 1709708 1412457 369565 58246 122479 cytidine N/F N/F 2079829 1907198 1302761 3073133 3278533 3975985 2560971 2804799 2576579 594651 73350 146693 cytosine N/F N/F N/F 407066 295881 657932 543958 533579 598247 721765 411270 212575 N/F N/F dATP N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F dCTP N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F dCytidine N/F N/F 3926235 4731185 3198738 573884 471482 269840 2358836 2274125 2253845 644618 158774 132349 dihydroorotate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F dihydroxyacetone_phosphate N/F N/F 3168527 2252278 1788568 287191 383372 451837 1306994 1141511 1332380 163824 N/F N/F dimethylglicine N/F N/F 141246579 143029290 153023203 8659594 8697723 10628836 76740418 74391519 75847004 20333407 6069783 6215254 dTMP N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F dTTP N/F N/F 1463098 1145980 1734219 2255625 2270136 2089641 1571478 1816101 1786081 1332144 307358 305846 dUMP N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F erythrose_4-phosphate N/F N/F 408959 634435 404388 N/F N/F N/F 208032 160491 159640 N/F N/F N/F F6P_G1P N/F N/F 7777109 10771834 6163167 1181020 1186164 1073419 4630593 4748294 4663590 1356859 346740 430435 FAD N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F 425747 folic_acid N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F formyl_methionine N/F N/F 641825 653512 508221 390404 487765 588072 586170 579077 602303 100393 N/F N/F "Fructose 1,6-bisphosphate" N/F N/F 1538694 731406 634457 1565316 1229254 1152882 1120386 1142131 1142194 144465 N/F N/F fructose N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F fumarate N/F N/F 32373951 33463213 15892651 17818710 15440022 13044084 21708159 22012723 21790132 7801747 1979048 1929355 gamma-aminobutyric_acid N/F N/F 20499073 18096654 13997224 23145609 13842522 15084765 16798998 18320601 16681061 7132788 N/F N/F gamma-glutamyl-alanine N/F N/F 1089127 528249 579778 3715759 4903676 3065927 1854405 1910181 2208171 687879 40242 127099 GAR N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F GDP N/F N/F 818906 630925 975430 856401 672472 789736 753659 808674 789564 186058 36793 53060 Glucose 6-phosphate 21743 N/F 9000009 11957071 8118873 1617082 1518884 1523497 5583378 5445050 5513610 1791244 555404 592127 glucose N/F N/F 65545501 87858011 54473499 1522150 1423240 1611719 41165773 37839828 39108717 24672789 11851060 13483649 "glutamate-13C5,15N" N/F N/F 488113 457282 716897 531368 620301 623185 555757 457033 502848 188693 N/F N/F glutamate N/F 56543 182025441 157330969 139903177 453030520 471714291 470873608 300776379 297516807 300602173 134753200 33414728 37047387 glutamine N/F N/F 3130556556 3069249833 2525628416 484975335 410660079 451874819 1762515497 1748549969 1761843721 591179280 177073173 173848545 glyceraldehyde_3-phosphate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F glycerate N/F N/F 105829965 86249978 107341849 113880666 104005287 103896860 104912750 101396397 97720482 36466827 11938148 12086080 Glycerol 3-Phosphate N/F N/F 94985395 116868493 86016517 17772899 20453538 19013093 63458121 60517645 62022863 23280739 7308696 7937414 Glycerophosphocholine N/F 280348 4863082930 5801223913 3605247077 17364699853 13160615621 23632369980 13788186413 13963709376 14030679980 3889116365 1044809611 1092538325 glycine N/F N/F 9898159 9874091 8756545 9373090 9377506 9537676 9039721 9020240 8933011 2758122 797138 847953 glycine_13C2-15N N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F GMP 196432 N/F 20999451 20500314 15917883 13124708 12268430 13515189 15857080 15920379 15662919 5090769 1545255 1541596 GSH-13C2_15N N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F GSH N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F GSH_13C2-15N-Ellmans N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F GSH_Ellmans N/F N/F 540904607 543854892 430524004 55388538 93687454 118071538 290701171 292057153 298932672 54033735 8438546 9557381 GSSG 61522 N/F 39161990 70732822 29275115 13859758 4057710 6277185 22565069 24218517 23772323 7052983 1944452 1911482 GTP N/F N/F 123509 54247 62409 N/F N/F N/F N/F 53025 67220 N/F N/F N/F guanidinoacetate N/F N/F 42120260 36258262 23129007 337517936 355173529 353169575 190774082 186763129 188606400 62052376 15247770 15730836 guanine N/F N/F 3351227 3318383 1737716 14967260 13464671 13465733 7868468 8017481 7675350 2725726 838560 856233 guanosine N/F N/F 11316171 14219458 6935653 19824559 21839424 12226625 13686252 13915144 13816156 3766429 1068592 1134044 histidine-13C6-15N3 N/F N/F 10766590 10912025 13710420 17553154 16899560 16382108 14659898 14488270 13938774 4650143 950391 923988 histidine 130205 N/F 1426535116 1177126778 1112313280 277573460 258935591 243229346 863978663 808670589 736451761 214811525 42094237 59451176 homocitrulline N/F N/F 4435976 4583038 4313078 6438521 7429251 5278111 6543998 5266916 5140969 1513557 232627 285136 homocysteine N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F hydroxyproline_gammaALA N/F N/F 1969106 3041873 1543181 2369223 2603714 2539637 2437806 2437022 2388331 897089 428561 391723 hypotaurine N/F N/F 2635315 5643021 3226391 996441 1604974 1411424 2291054 2492824 2513231 630849 139504 128648 hypoxanthine N/F N/F 670877022 1089500180 720653542 1911730616 1909124196 2035148114 1409611712 1425954345 1414186662 527490106 178443518 184620299 IMP N/F N/F 38758684 49980760 44529755 52756733 57333082 67332497 51060431 50971222 52278266 15412825 4560910 4804151 indolelactate N/F N/F N/F N/F N/F N/F N/F N/F N/F 121069 N/F N/F N/F N/F inosine N/F N/F 257604014 397014396 283948053 292009239 300793507 240529842 292333899 283979693 287051308 112376738 37574362 41955629 isocitrate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Isoleucine-13C6-15N N/F N/F 2975137 1490939 1875157 2377877 2371830 2459592 1793798 1965701 2629705 560355 213665 188385 isoleucine N/F N/F 70566749 70367167 67018861 26903381 25157382 27581030 46098937 46690458 45839288 13050243 3051800 3401971 Itaconic Acid N/F N/F 188723758 167439566 178475478 177314097 171784384 166532316 161223030 169024875 180727557 75690995 27823800 28450883 Kynurenic Acid N/F N/F 809356 388626 1153009 1117957 562544 2319586 989972 677935 1114039 376405 N/F N/F kynurenine N/F N/F 3085352 4666719 2402022 394120 216941 269533 1747188 1761643 1739449 597822 190825 206954 lactic_acid N/F N/F 1216232185 1531613704 1030377613 531961309 493938668 526551986 874939155 875615982 878256939 263069306 69179919 83227113 leucine-13C6-15N N/F N/F 2679031 2587739 3008460 2688402 2700496 2310673 2762635 2929080 2724410 887802 174919 273995 leucine N/F N/F 112399347 106980931 98412510 42317252 36766183 38047945 72028543 69702806 73167396 19882078 5202417 6168047 lysine-13C6-15N2 N/F N/F 1164595 1002591 1212193 1489054 1404733 1305409 906741 1283053 1301798 332989 N/F N/F lysine N/F N/F 162883060 163328031 143752293 45419063 45817206 36378203 87382905 97736584 102460970 32083688 11576509 10416095 malate N/F N/F 316341862 333203444 167588484 177137802 163763220 138576568 208221599 218062941 221977896 89836025 23311403 27067827 mannose N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Methionine sulfoxide N/F N/F 5357455 3518138 2973147 7452907 7902614 8300451 5601732 5775526 5997164 1120023 243393 342049 methionine-13C5-15N N/F N/F 3368999 2860278 3936315 4512317 3930810 3741931 3117144 3769193 3717336 1122602 321406 340819 methionine N/F N/F 56700692 48348738 41576132 75010327 72890298 69518532 60108479 60851695 63073853 20690687 6499625 6797765 methylthioadenosine N/F N/F 1243040 1611847 1092329 1056599 1242369 1039690 1164146 1148611 1256425 312309 N/F N/F myo-inositol N/F N/F 11665601 9045061 10197709 196197419 176754706 234259620 94489563 93814428 97922758 60750689 34668083 32795625 N-acetylcysteine N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N-acetylglutamate N/F N/F 464750 626044 33820821 535144 583778 501529 510924 889602 966671 200636 44255 82516 N-acetylglutamine N/F N/F 119372900 108452695 82768901 7873166 7217494 8355096 57057096 58059897 54570917 17820226 5407004 5579200 N-acetyllysine N/F N/F 9411471 11011479 9389793 9281252 7735162 6414670 8221193 8257811 8332404 3541142 629175 611810 N-acetylmethionine N/F N/F 285725 494460 199448 234979 147992 136293 281806 232105 242995 N/F N/F N/F N-acetylornithine N/F N/F 1127219 1560779 2020882 N/F 236175 233345 1143588 831384 705510 396058 N/F N/F N-acetylphenylalanine N/F N/F 2095069 2077376 1842414 1089406 1218948 1109055 1419770 1650914 1609702 611494 231679 171308 NAD+ N/F N/F 225500171 222699768 264115212 183276648 201250454 218277944 218702758 221120338 222573175 68748843 20154171 19090862 NADH N/F N/F 74361289 58968648 120411837 13404821 15937577 13539138 42400299 45854974 40447754 14472675 4190130 4548512 NADP N/F N/F 25981734 18253308 18911802 13593152 13023689 15349471 16531876 16320117 17007776 5150005 1367281 1273399 NADPH N/F N/F 2854894 70128 7907461 930848 1249629 1189142 2243076 2362963 2031488 628171 47075 20731 niacinamide N/F N/F 3012766078 4235931229 1361782833 1249127383 1121592062 856018977 2069231426 2011739868 2077783794 666802018 207668661 209143391 Nicotinic Acid N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F O-phosphoethanolamine N/F N/F 16918164 15524393 13041466 56532846 58053074 63978068 35864010 35924670 36036588 12051371 3448143 3953788 ornithine 899713 626422 73235716 74101860 57191247 11548981 10243655 10931730 38190782 43148259 43435485 16896529 5785827 6334093 orotate N/F N/F 3316520 2795764 2617508 3407662 2983786 3380113 3056165 2954824 2974007 1013504 277265 412476 p-aminobenzoate N/F N/F 1278196 1574744 1747204 1611569 1861718 2326437 1685830 1863528 1843115 610990 204224 268456 palmitate N/F N/F 21643898 33388375 15440231 24876995 28555686 23693204 N/F 18893753 23858962 6971435 2198863 N/F Pantothenate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Phenylacetyl glutamine N/F N/F 355151 473241 50281 515894 574229 113668 262809 296475 340104 45194 N/F N/F phenylalanine-13C9-15N N/F N/F 9512755 7708235 9675324 11327617 10738230 9176416 10021572 9897124 9781293 2356920 639042 636939 phenylalanine N/F N/F 42581848 42010245 36461055 18499957 16993271 18550183 29643874 28497863 28921709 9699788 3695362 4109878 phosphocholine N/F N/F 1580744057 2938647574 1845180764 2399220407 2458246414 2752475192 2215205271 2245999288 2285992710 490895811 102302357 107378116 phosphocreatine N/F N/F 2829137 2777525 2547012 2193756 2575915 1718032 2246603 2402655 2338970 736393 200391 227814 phosphoenolpyruvate N/F N/F 1642657 1788365 834420 1362947 808830 594470 1433843 1046795 1057190 98464 N/F N/F phosphoethanolamine N/F N/F 15631874 14619495 12527752 52940265 52210794 57157582 34089349 32302005 33976635 9731588 2820878 3055995 phosphoserine N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Picolinate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Pipecolate N/F N/F 10377414 11482522 8476080 10772894 7733394 8251678 8789159 8882496 9932625 2476913 873414 822496 proline-13C5-15N N/F N/F 12653856 11469871 13084077 13709129 13318033 11982859 12062098 12475899 12371463 3642930 1026573 1064140 proline N/F N/F 778795175 617557742 526241898 202567024 169986170 186935430 397729302 410397892 390993859 112238209 30866432 34267387 propionylcarnitine N/F N/F 349112415 346024143 317100900 95172019 80101826 98822883 215507743 211084447 206880283 67980992 18505129 21702359 PRPP N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F pseudouridine N/F N/F 1582464 1939558 1213159 1750369 1506970 2208718 1674842 1778648 1612521 800227 296498 296142 Pyridoxal N/F N/F 1469525 1991034 1065683 3206227 2557622 2313927 1753362 1902460 2017499 557119 N/F N/F Pyridoxine N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F pyruvate N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Riboflavin N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F Ribose 1-phosphate N/F N/F 1385638 1262930 1272715 729393 783025 718275 1086627 1120551 998189 230361 68937 73880 Ribose_Ribulose 5-phosphate_3Me-Lys N/F N/F 1524752 1497179 1527645 729393 783025 718275 1086627 1120551 998189 294862 68937 78841 S-Adenosyl-L-Homocysteine N/F N/F 330281746 325998578 313509318 54132379 56617807 61717806 187705560 203750168 202206063 48377829 13213936 12846227 S-Adenosyl-L-methionine N/F N/F 185236972 218726429 143823428 187397009 181634993 179519164 169553647 174387161 177618014 60641546 14249379 13452927 S-methylcysteine N/F N/F 17446 N/F N/F 72516 146152 43071 32526 71325 52665 N/F N/F N/F saccharopine N/F N/F 48719288 49673039 36126610 4456531 4694055 3418584 24199600 25669523 26652747 1923099 314338 429099 sarcosine N/F N/F 50266215 44409304 43194562 912267 932593 383496 23342324 23384233 25380672 5141950 1156159 1251835 Sedoheptulose-7-phosphate N/F N/F 6734459 9004880 3688431 1413806 1606204 1869938 3993073 3969507 4020455 1085928 304647 284066 serine-13C3-15N N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F serine N/F N/F 8385139 7527369 5004586 6082779 4991482 5677610 5849088 6205050 6043569 1926688 487028 493227 sorbitol N/F N/F 3701416 8319482 3599776 43201409 3025354 289213915 57691602 58090717 55354971 24851317 12213211 12329053 succinate N/F N/F 90425889 107290263 76250571 42644050 37782634 46454087 68421402 62849006 64505956 17345163 5107853 5254933 taurine N/F N/F 1663524934 1891993825 1851410023 793294740 669317906 828874245 1319732610 1281438100 1305075517 688735552 340315576 338650219 thiamine N/F N/F 13363 20671 11979 33301 38224 46159 32260 35544 24565 N/F N/F N/F threonine-13C4-15N N/F N/F 799586 851348 689144 245242 161326 104116 410830 293607 424749 N/F N/F 34974 threonine N/F N/F 60875672 52119407 41067201 17155912 15109081 16581733 28695027 30561489 26278826 8155348 2034581 2575544 thymidine N/F N/F 17194 23158 N/F 130098 102153 58322 59865 70628 59139 25064 N/F N/F trans-urocanate N/F N/F 8484480 1938677 3505867 2376716 6317304 3563266 4470231 4376723 4572582 1135731 N/F N/F tryptophan N/F N/F 54100899 48445723 46219196 33581386 28205874 35837443 41186442 41598263 41619658 12938352 4117047 4472880 tyrosine-13C9-15N N/F N/F 3261372 2807708 3353463 3544096 3318170 3096487 3105505 3233452 3083128 905231 195128 159441 tyrosine N/F N/F 107946498 92041935 73583985 58389700 48864857 46854821 69413456 70989623 72781864 20990812 6746079 6394529 UDP-GlcNAc N/F N/F 34651262 39908374 25939324 15253534 16642170 15446544 23720724 24446406 24926649 7769145 2358664 2384813 UDP-hexose N/F N/F 37408284 48535521 25048469 15392888 15485447 14568321 24927493 24498324 24655510 6833957 1982170 2060546 UDP N/F N/F 1942090 2841891 2012589 556622 535985 588784 1351833 1316293 1380584 350104 81113 76683 UMP N/F N/F 38125685 52288268 32481658 7232319 7917860 7873548 23705386 23327345 23416928 6829991 1897887 2139165 uracil N/F N/F 1211853 1978790 779494 13399668 13891842 14974136 8221557 7350157 8279653 3076375 1052520 1751660 Ureidosuccinic_acid N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F N/F uric acid N/F N/F 131841808 171989418 144666029 36181522 33309415 36017682 98159794 98421710 100791746 46891327 16634188 16834334 uridine N/F N/F 2740797 5671958 2252386 1501739 1607522 1466942 2263351 2224034 2199364 1148858 395701 418011 UTP N/F N/F 640407 722119 455626 48047 79086 53043 335393 291880 302169 36862 N/F N/F valine-13C5-15N N/F N/F 1528451 1307986 1414563 1481863 1432273 1334924 1649382 1399471 1502426 318804 91554 N/F valine N/F N/F 74070012 66146383 72058426 34750060 29769600 32328253 49917011 52279628 52891807 15967878 N/F N/F xanthine N/F 184862 205193966 332578617 248768634 188829350 188380396 206264402 222757027 228325585 231165673 133667305 61244145 62611583 xanthosine N/F N/F 21391297 31058947 23269271 1605583 1434871 1513249 13660210 13990471 14051931 5549994 1871316 1919826 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 1-methylnicotinamide "2,3-BPG" 2-aminobutyric_acid 2-hydroxybutyric_acid 2-hydroxyglutarate 2-oxoadipate 2-oxobutyric_acid 3-hydroxybutyric_acid 3-hydroxyisobutyric_acid 3-methyl-2-oxopentanoic_acid 3-phosphoglycerate 3-ureidopropionate 5-Hydroxyindole-3-Acetate 6-phosphogluconate acetoacetate Acetyl CoA acetylalanine acetylaspartate acetylcarnitine acetylglutamate acetylglycine acetylmethionine acetylserine adenine adenosine ADP-ribose ADP AICAR alanine-13C3-15N alanine allantoin alpha-keto-isovalerate Alpha-Ketoglutarate Alpha-Lipoic Acid amidoadipate AMP anserine arginine-13C6-15N4 arginine argininosuccinate ascorbate asparagine aspartate-13C6-15N4 aspartate asymmetric dimethylarginine (ADMA) ATP beta-alanine betaine biotin Butyrylcarnitine C5 Carnitines cAMP carnitine carnosine CDP-choline CDP choline cis-aconitate citrate citrulline CMP CoA-Ellmans Coenzyme A creatine creatinine CTP cystathionine cysteic_acid cysteine-glycine cysteine_13C3_15N1 cysteine cystine cytidine cytosine dATP dCTP dCytidine dihydroorotate dihydroxyacetone_phosphate dimethylglicine dTMP dTTP dUMP erythrose_4-phosphate F6P_G1P FAD folic_acid formyl_methionine "Fructose 1,6-bisphosphate" fructose fumarate gamma-aminobutyric_acid gamma-glutamyl-alanine GAR GDP Glucose 6-phosphate glucose "glutamate-13C5,15N" glutamate glutamine glyceraldehyde_3-phosphate glycerate Glycerol 3-Phosphate Glycerophosphocholine glycine glycine_13C2-15N GMP GSH-13C2_15N GSH GSH_13C2-15N-Ellmans GSH_Ellmans GSSG GTP guanidinoacetate guanine guanosine histidine-13C6-15N3 histidine homocitrulline homocysteine hydroxyproline_gammaALA hypotaurine hypoxanthine IMP indolelactate inosine isocitrate Isoleucine-13C6-15N isoleucine Itaconic Acid Kynurenic Acid kynurenine lactic_acid leucine-13C6-15N leucine lysine-13C6-15N2 lysine malate mannose Methionine sulfoxide methionine-13C5-15N methionine methylthioadenosine myo-inositol N-acetylcysteine N-acetylglutamate N-acetylglutamine N-acetyllysine N-acetylmethionine N-acetylornithine N-acetylphenylalanine NAD+ NADH NADP NADPH niacinamide Nicotinic Acid O-phosphoethanolamine ornithine orotate p-aminobenzoate palmitate Pantothenate Phenylacetyl glutamine phenylalanine-13C9-15N phenylalanine phosphocholine phosphocreatine phosphoenolpyruvate phosphoethanolamine phosphoserine Picolinate Pipecolate proline-13C5-15N proline propionylcarnitine PRPP pseudouridine Pyridoxal Pyridoxine pyruvate Riboflavin Ribose 1-phosphate Ribose_Ribulose 5-phosphate_3Me-Lys S-Adenosyl-L-Homocysteine S-Adenosyl-L-methionine S-methylcysteine saccharopine sarcosine Sedoheptulose-7-phosphate serine-13C3-15N serine sorbitol succinate taurine thiamine threonine-13C4-15N threonine thymidine trans-urocanate tryptophan tyrosine-13C9-15N tyrosine UDP-GlcNAc UDP-hexose UDP UMP uracil Ureidosuccinic_acid uric acid uridine UTP valine-13C5-15N valine xanthine xanthosine METABOLITES_END #END