#METABOLOMICS WORKBENCH SIMR_Core_Facility_20231129_202258 DATATRACK_ID:4483 STUDY_ID:ST003029 ANALYSIS_ID:AN004966 PROJECT_ID:PR001882 VERSION 1 CREATED_ON December 27, 2023, 1:41 am #PROJECT PR:PROJECT_TITLE Estrogen-mediated inhibition of purine metabolism and cell cycle arrest as a PR:PROJECT_TITLE novel therapeutic approach in colorectal cancer cells PR:PROJECT_SUMMARY Purine metabolism is upregulated in various cancers including colorectal cancer PR:PROJECT_SUMMARY (CRC). While previous research has elucidated the role of Estrogen (E2) in PR:PROJECT_SUMMARY metabolism remodeling and ATP production, its effects on purine metabolism PR:PROJECT_SUMMARY remained unexplored. This study investigates the impact of E2 signalling on PR:PROJECT_SUMMARY purine metabolism in CRC cells. We demonstrate, for the first time, a protective PR:PROJECT_SUMMARY effect of E2 on CRC cells by targeting the purine synthesis pathway through its PR:PROJECT_SUMMARY receptor estrogen receptor α (ERα). A full metabolomic profiling, next PR:PROJECT_SUMMARY generation sequencing (NGS) and integrated OMICS were conducted for HCT-116 PR:PROJECT_SUMMARY cells treated with E2 with and without silencing ERα. Our results revealed an PR:PROJECT_SUMMARY enrichment of the purine metabolic pathway, with 27 genes in the de novo purine PR:PROJECT_SUMMARY synthesis pathway downregulated in E2-treated CRC cells. Besides, E2-induced DNA PR:PROJECT_SUMMARY damage, cell cycle arrest, and apoptosis are ERα-dependent. Our findings PR:PROJECT_SUMMARY suggest potential therapeutic avenues for CRC treatment through antimetabolites PR:PROJECT_SUMMARY targeting purine synthesis, as E2 treatment reduces the expression of relevant PR:PROJECT_SUMMARY metabolites. PR:INSTITUTE Sharjah Institute for Medical Research PR:LAST_NAME Facility PR:FIRST_NAME Core PR:ADDRESS M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah PR:EMAIL tims-tof@sharjah.ac.ae PR:PHONE 065057656 #STUDY ST:STUDY_TITLE Estrogen-mediated inhibition of purine metabolism and cell cycle arrest as a ST:STUDY_TITLE novel therapeutic approach in colorectal cancer cells ST:STUDY_SUMMARY Purine metabolism is upregulated in various cancers including colorectal cancer ST:STUDY_SUMMARY (CRC). While previous research has elucidated the role of Estrogen (E2) in ST:STUDY_SUMMARY metabolism remodeling and ATP production, its effects on purine metabolism ST:STUDY_SUMMARY remained unexplored. This study investigates the impact of E2 signalling on ST:STUDY_SUMMARY purine metabolism in CRC cells. We demonstrate, for the first time, a protective ST:STUDY_SUMMARY effect of E2 on CRC cells by targeting the purine synthesis pathway through its ST:STUDY_SUMMARY receptor estrogen receptor α (ERα). A full metabolomic profiling, next ST:STUDY_SUMMARY generation sequencing (NGS) and integrated OMICS were conducted for HCT-116 ST:STUDY_SUMMARY cells treated with E2 with and without silencing ERα. Our results revealed an ST:STUDY_SUMMARY enrichment of the purine metabolic pathway, with 27 genes in the de novo purine ST:STUDY_SUMMARY synthesis pathway downregulated in E2-treated CRC cells. Besides, E2-induced DNA ST:STUDY_SUMMARY damage, cell cycle arrest, and apoptosis are ERα-dependent. Our findings ST:STUDY_SUMMARY suggest potential therapeutic avenues for CRC treatment through antimetabolites ST:STUDY_SUMMARY targeting purine synthesis, as E2 treatment reduces the expression of relevant ST:STUDY_SUMMARY metabolites. ST:INSTITUTE Sharjah Institute for Medical Research ST:LAST_NAME Facility ST:FIRST_NAME Core ST:ADDRESS M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, ST:ADDRESS UAE, Sharjah, 000, United Arab Emirates ST:EMAIL tims-tof@sharjah.ac.ae ST:PHONE +971 6 5057656 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Colorectal cancer cells #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 - control_1-01-8852 Treatment:Control RAW_FILE_NAME=control_1-01-8852.d SUBJECT_SAMPLE_FACTORS - control_1-02-8853 Treatment:Control RAW_FILE_NAME=control_1-02-8853.d SUBJECT_SAMPLE_FACTORS - control_2-01-8854 Treatment:Control RAW_FILE_NAME=control_2-01-8854.d SUBJECT_SAMPLE_FACTORS - control_2-02-8855 Treatment:Control RAW_FILE_NAME=control_2-02-8855.d SUBJECT_SAMPLE_FACTORS - control_3-01-8856 Treatment:Control RAW_FILE_NAME=control_3-01-8856.d SUBJECT_SAMPLE_FACTORS - control_3-02-8857 Treatment:Control RAW_FILE_NAME=control_3-02-8857.d SUBJECT_SAMPLE_FACTORS - E2_1-01-8860 Treatment:17-β-estradiol RAW_FILE_NAME=E2_1-01-8860.d SUBJECT_SAMPLE_FACTORS - E2_1-02-8861 Treatment:17-β-estradiol RAW_FILE_NAME=E2_1-02-8861.d SUBJECT_SAMPLE_FACTORS - E2_2-01-8862 Treatment:17-β-estradiol RAW_FILE_NAME=E2_2-01-8862.d SUBJECT_SAMPLE_FACTORS - E2_2-02-8863 Treatment:17-β-estradiol RAW_FILE_NAME=E2_2-02-8863.d SUBJECT_SAMPLE_FACTORS - E2_3-01-8864 Treatment:17-β-estradiol RAW_FILE_NAME=E2_3-01-8864.d SUBJECT_SAMPLE_FACTORS - E2_3-02-8865 Treatment:17-β-estradiol RAW_FILE_NAME=E2_3-02-8865.d #COLLECTION CO:COLLECTION_SUMMARY HCT-116 cells were cultured using a complete RPMI-1640 medium (Sigma-Aldrich, CO:COLLECTION_SUMMARY St. Louis, MO, USA), supplemented with 2 mM l-glutamine, 1% non-essential amino CO:COLLECTION_SUMMARY acids, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum. CO:COLLECTION_SUMMARY Depending on each experimental run, HCT-116 cells were seeded and incubated at CO:COLLECTION_SUMMARY 37°C in a 5% CO2 environment for 24 hours CO:SAMPLE_TYPE Colerectal cancer cells #TREATMENT TR:TREATMENT_SUMMARY 20 nM 17-β-estradiol (E2), diluted in 70% ethanol, (Sigma-Aldrich, St. Louis, TR:TREATMENT_SUMMARY MO, USA), was introduced to the cells for 48 hours. Cells designated as control TR:TREATMENT_SUMMARY received the same volume of 70% ethanol, serving as the vehicle. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To assess the intracellular metabolites, whole metabolites levels were measured SP:SAMPLEPREP_SUMMARY after proper treatment. In brief, 1.0 × 106 of HCT-116 cells were collected and SP:SAMPLEPREP_SUMMARY cell extracts were washed twice with 5% of mannitol. The pellet was lysed using SP:SAMPLEPREP_SUMMARY 400 uL of protease inhibitor dissolved in lysis buffer, allowing it to sit for SP:SAMPLEPREP_SUMMARY 10 minutes at room temperature. Subsequently, all samples underwent 30 seconds SP:SAMPLEPREP_SUMMARY of vortexing and were sonicated using either the COPLEY sonicator or QSONICA SP:SAMPLEPREP_SUMMARY SONICATOR (Qsonica, Newtown, CT, USA) at 30% AMP for 30 seconds until the SP:SAMPLEPREP_SUMMARY pellets dissolved. The resulting solution was transferred to new Eppendorf tubes SP:SAMPLEPREP_SUMMARY and centrifuged at 14000 rpm for 5 minutes. After centrifugation, the SP:SAMPLEPREP_SUMMARY supernatant layer was carefully moved to another Eppendorf tube, to which 400 uL SP:SAMPLEPREP_SUMMARY of methanol and 300 uL of chloroform were added. Each mixture was vortexed for SP:SAMPLEPREP_SUMMARY 30 seconds and then centrifuged at 14000 rpm for 5 minutes. The upper layer was SP:SAMPLEPREP_SUMMARY transferred and stored in a glass vial. For washing, 300 uL of methanol was SP:SAMPLEPREP_SUMMARY added to the white disk and lower layer, vortexed until the white disk broke and SP:SAMPLEPREP_SUMMARY settled at the bottom of the Eppendorf. Subsequently, samples were centrifuged SP:SAMPLEPREP_SUMMARY at 14000 rpm for 3 minutes, and the supernatant was transferred into the same SP:SAMPLEPREP_SUMMARY glass vial. To dry the solvent, the samples were processed in the EZ-2 Plus SP:SAMPLEPREP_SUMMARY (GeneVac, Ipswich, UK) at 37 ± 1 °C. The dried samples were resuspended with SP:SAMPLEPREP_SUMMARY 200 µL (0.1% formic acid in deionized water) and vortexed for 2 minutes for SP:SAMPLEPREP_SUMMARY thorough mixing. Finally, the samples were filtered using a hydrophilic nylon SP:SAMPLEPREP_SUMMARY syringe filter with a 0.45 µm pore size and returned to the glass insert within SP:SAMPLEPREP_SUMMARY LC glass vials for analysis by Q-TOF MS. A quality control (QC) sample was SP:SAMPLEPREP_SUMMARY prepared by pooling the same volume (10 uL) from each sample, and all samples SP:SAMPLEPREP_SUMMARY were placed in the autosampler at a temperature set at 4℃. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY An ultra-high-performance liquid chromatography system; Elute UHPLC (Bruker CH:CHROMATOGRAPHY_SUMMARY Daltonik GmbH, Bremen, Germany) . 10 µL aliquot of the sample was injected and CH:CHROMATOGRAPHY_SUMMARY the separation was performed on a Hamilton® Intensity Solo C18 column (2.1 × CH:CHROMATOGRAPHY_SUMMARY 100 mm, 1.8 µm) (Bruker Daltonik) in a column oven temperature set at 35 ◦C , CH:CHROMATOGRAPHY_SUMMARY using solvent A (0.1% formic acid in deionized Water) and solvent B (0.1% formic CH:CHROMATOGRAPHY_SUMMARY acid in acetonitrile) with the following gradient elution mode: 0 to 2 min, 1% CH:CHROMATOGRAPHY_SUMMARY B; 2 to 17 min, 1–99% B; 17 to 20 min, 99% B; 20 to 20.1 min, 99–1% B; 20.1 CH:CHROMATOGRAPHY_SUMMARY to 30 min, 1% B. The flow rate was 0.25 mL/min from 0 to 20 min, 0.35 mL/min CH:CHROMATOGRAPHY_SUMMARY from 20 min to 28.3 min, and 0.25 mL/min from 28.3 to 30 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Bruker Elute CH:COLUMN_NAME Hamilton® Intensity Solo 2 C18 column (2.1 × 100 mm, 1.8 µm) CH:SOLVENT_A Water (0.1% Formic Acid) CH:SOLVENT_B ACN (0.1% Formic Acid) CH:FLOW_GRADIENT gradient elution mode: 0 to 2 min, 1% B; 2 to 17 min, 1–99% B; 17 to 20 min, CH:FLOW_GRADIENT 99% B; 20 to 20.1 min, 99–1% B; 20.1 to 30 min, 1% B. CH:FLOW_RATE The flow rate was 0.25 mL/min from 0 to 20 min, 0.35 mL/min from 20 min to 28.3 CH:FLOW_RATE min, and 0.25 mL/min from 28.3 to 30 min. CH:COLUMN_TEMPERATURE 35 ◦C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker timsTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The ESI source condition for every injection was as follows: The drying gas flow MS:MS_COMMENTS rate was 10.0 L/min at a temp of 220 ◦C; the capillary voltage was set at 4500 MS:MS_COMMENTS V; the End Plate offset was set at 500 V; the nebulizer pressure of 2.2 bar. The MS:MS_COMMENTS acquisition involved two segments; auto MS scan, which ranged from 0 to 0.3 min MS:MS_COMMENTS for the calibrant sodium formate, and auto MS/MS scan with CID acquisition, MS:MS_COMMENTS which included fragmentation and ranged from 0.3 to 30 min. The acquisition in MS:MS_COMMENTS both segments was performed using the positive mode at 12 Hz. The automatic MS:MS_COMMENTS in-run mass scan range was from 50 to 1300 m/z, the width of the precursor ion MS:MS_COMMENTS was ±0.5, the cycle time was 0.5 sec, and the threshold was 400 cts. Active MS:MS_COMMENTS exclusion was excluded after 3 spectra and released after 0.2 min. For MS2 MS:MS_COMMENTS acquisition the data dependent acquisition (DDA) was used, and the collision MS:MS_COMMENTS energy stepping fluctuated between 100 and 250% set at 20 eV. M/Z measurements MS:MS_COMMENTS were externally calibrated using 10 mM of sodium formate before sample analysis. MS:MS_COMMENTS In addition, sodium formate solution was injected at the beginning of each MS:MS_COMMENTS sample run and used for internal calibration during data processing MS:MS_COMMENTS TRX-2101/RT-28-calibrants for Bruker T-ReX LC-QTOF (Nova Medical Testing Inc.) MS:MS_COMMENTS was injected before sample analysis to check and test the performance of the MS:MS_COMMENTS column, reversed-phase liquid chromatography (RPLC) separation, multipoint MS:MS_COMMENTS retention time calibration, and the mass spectrometer. Also, TRX-3112-R/MS MS:MS_COMMENTS Certified Human serum for Bruker T-ReX LC-QTOF solution (Nova Medical Testing MS:MS_COMMENTS Inc.) was prepared from pooled human blood and injected before sample analysis MS:MS_COMMENTS to check the performance of the LC-MS instruments. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples control_1-01-8852 control_1-02-8853 control_2-01-8854 control_2-02-8855 control_3-01-8856 control_3-02-8857 E2_1-01-8860 E2_1-02-8861 E2_2-01-8862 E2_2-02-8863 E2_3-01-8864 E2_3-02-8865 Factors Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:17-β-estradiol Treatment:17-β-estradiol Treatment:17-β-estradiol Treatment:17-β-estradiol Treatment:17-β-estradiol Treatment:17-β-estradiol 1-Methyladenosine 1792 1340 1422 878 1192 1092 692 706 1144 748 470 400 17-Hydroxyprogesterone 4704 5764 3402 4312 8938 7568 6814 7974 3112 5258 2214 10110 2'-Deoxyguanosine 5'-monophosphate 2544 1978 3066 2708 3664 3624 2416 2524 3494 3616 3064 3608 2,4-Diaminobutyric acid 29300 26700 51202 56194 15308 13204 14296 12258 33566 28536 53766 50140 2-Ethyl-2-hydroxybutyric acid 54634 51002 48736 52232 51790 53312 37078 35918 47728 45742 30236 28900 2-Hydroxyadenine 42140 47800 58240 55158 44602 46120 36540 37378 15726 14138 38084 38004 2-Phenylaminoadenosine 114630 98216 135362 95490 88502 80058 45012 48366 92588 73972 47710 45360 2-Phenylbutyric acid 60676 60586 60924 58712 61314 57832 56260 57248 59266 57020 58922 51314 2-Pyrrolidinone 10586 11184 12840 9080 9040 11232 9176 8776 11254 10832 10546 10018 3,4-Dihydroxyphenylglycol 1348 1058 1250 906 994 1158 938 974 1142 992 668 796 3-Hydroxy-4-methoxycinnamic aicd 5870 6084 6202 4810 4808 5686 4620 4608 4706 4292 4914 5020 3-Methylindole 4200 3758 4666 4196 3490 3228 4422 4334 3878 3278 4066 3558 3a,6b,7b-Trihydroxy-5b-cholanoic acid 253438 247570 256244 237640 256832 236168 289790 287014 270006 280844 265954 276420 5'-Methylthioadenosine 98110 95468 109700 107952 101042 104790 110326 104144 111126 109818 105850 88100 5-Aminopentanoic acid 3740 2912 4098 3368 2150 2438 4182 3296 3308 3966 3238 2622 5-Methoxysalicylic acid 27922 28946 36114 24706 28838 26732 20360 23222 33984 25878 17528 15562 5-Methoxytryptophol 3156 2986 2924 2402 2332 2612 718 724 1692 1582 844 858 7,8-Dihydrobiopterin 650 838 1442 1516 1362 2074 1050 1360 1484 2038 1302 1028 Acetoin 349224 308798 407986 324356 219948 207578 179298 175446 384658 309040 412804 202184 Adenine 93420 96096 91812 97126 78258 87466 74956 70612 100330 103244 90572 85946 Adenosine 188028 180662 185630 197824 179958 159476 153044 154300 201464 215530 181878 164412 Adenosine 3',5'-diphosphate 38094 37354 36854 35518 37976 37322 19784 20764 26604 26816 33412 34132 Adenosine monophosphate 3450 4838 3754 9678 9008 9702 11892 10530 8630 6904 6116 9790 Adenosine triphosphate 3366 2498 1010 3220 3012 3564 1186 1362 1864 1556 3602 3052 Allocholic acid 7008 6616 7112 7276 4792 4234 3532 3942 6262 6726 6532 6022 Androstenedione 7132 8200 6310 6690 7872 6312 7150 6592 8230 10526 11702 10024 Aspartame 3856 3220 3464 3960 4330 2680 3678 3462 3586 3658 3636 3346 Aspirin 2870 2854 3328 3148 2810 2732 3558 3394 2954 2884 2896 2734 Atenolol 3046 1892 2679 2626 2590 2380 636 590 1876 1274 1624 1520 Benzoic acid 3970 3884 3884 3982 3888 3858 4572 4758 4138 3906 4714 4562 Carnosine 26988 18910 29906 23838 21584 24642 23952 23406 23352 23896 25430 24190 Cinnamaldehyde 18038 18046 18966 17188 16832 16990 15154 15457 19984 17610 18676 19050 Clotrimazole 1416 1766 1654 1864 2172 2342 1992 2134 1704 1842 2546 1942 Coumarin 2630 2362 2692 2992 2540 2662 2686 2838 2690 2590 2592 2576 Cyclic AMP 1634 1690 1532 1766 1924 2054 1014 1152 1538 1210 1320 1620 Cytosine 1482 1276 1772 1766 1442 1122 2432 2546 2148 2128 2292 2338 D-Glucurono-6,3-lactone 7800 8242 7656 8606 9220 8460 6662 7798 8252 8398 8960 8286 Diacetyl 11878 12306 12430 10512 11440 10370 6708 9846 9318 9406 12318 10088 Diethanolamine 1018 1272 1206 880 1052 1550 450 548 306 312 1484 576 Dopamine 1714 1688 1720 1202 1762 1818 1566 1564 2050 1854 1574 1834 Elaidic acid 8330 8244 8202 6928 7766 5534 4936 5340 4988 5108 6036 5390 Epinephrine 4150 5618 5286 5022 5578 6394 4468 4398 7258 6298 6480 5782 Folic acid 2684 2018 3426 2794 2242 2880 4768 4284 4520 4316 3260 2734 Galactitol 14818 15792 14156 15634 15836 14292 14580 16206 15284 15306 14160 14092 Gamma-Caprolactone 3052 3012 2992 3052 2354 2468 2316 2710 2370 2440 3044 3068 Gluconolactone 7078 12084 8054 20866 33682 32212 19298 18974 40010 40534 24746 21728 Glucosamine 610 598 384 542 588 576 424 416 0 0 0 0 Glutathione 32882 50074 39990 98554 134094 128832 82958 87824 179124 154850 101490 90748 Glycerophosphocholine 2988 2672 2950 2618 3720 3886 3614 4026 2928 2852 3662 3882 Guanidinosuccinic acid 39204 40296 33460 40236 24162 20754 35630 36572 54766 48546 47760 31308 Guanosine 25076 23742 22912 25756 22922 25762 20158 20662 7042 7590 19076 18364 Guanosine 5'-diphosphate 3502 3556 2022 2516 3254 3576 2784 2816 3410 2384 3124 2804 Guanosine monophosphate 2030 2010 1682 1800 2294 1970 2146 1702 1994 1980 1932 2070 Hydroxyisocaproic acid 0 0 249253 244366 205820 189848 177610 183584 275296 280802 142604 139808 Hyodeoxycholic acid 32806 34686 22988 30718 16218 14542 55682 57964 16766 22904 17614 26844 Hypoxanthine 208428 197474 228268 249928 225990 229914 179562 191810 213342 220792 207594 203876 Indole 24550 24312 29638 25746 22148 22128 27276 27214 27814 25358 25752 20150 Indoleacrylic acid 130402 134012 163218 147038 115838 116350 156778 157150 143190 142482 140828 112546 Inosine 106072 107714 94774 98158 93046 96910 73038 78902 82566 82750 80720 84382 Isobutyric acid 404650 340276 530622 390930 256926 241186 225082 222974 419316 348942 525200 217580 Isovalerylcarnitine 379606 405340 422288 392800 384286 370162 314368 288336 316798 334004 320290 311408 L-Acetylcarnitine 43388 43716 46470 39346 40034 39944 39906 44844 46804 45102 51992 45880 L-Fucose 2242 2676 2602 2600 2376 2104 2200 2240 2332 2102 2646 2452 L-Glutamic acid 712 1048 1090 1198 1202 1280 1330 878 1026 1226 1332 936 L-Leucine 16536 15764 16124 18026 14784 16558 18214 18516 16262 16226 15146 17416 L-Methionine 7530 8546 6008 7870 7798 7582 9794 9920 7622 7740 8432 10168 L-Norleucine 9318 9144 8078 7716 8016 8270 8784 8626 9824 9702 11448 11492 L-Phenylalanine 29842 32546 31642 28850 29146 27724 34424 35508 28176 31606 30406 28330 L-Proline 1370 1196 1616 1384 2008 1546 1464 1874 1308 2320 1746 1530 L-Thyronine 2090 2242 1946 2150 2514 2322 2174 2154 2116 2250 2254 2326 L-Tryptophan 34588 30358 39106 34084 28780 29116 37524 37752 34336 34212 33574 29124 L-Tyrosine 3906 3248 5148 5676 3284 3344 8502 9270 2620 2582 4620 4598 Leucinic acid 49150 51026 52012 46422 40700 38496 34840 34000 43796 44426 47380 48356 m-Coumaric acid 8498 9426 9438 9798 10424 9542 12678 10704 9896 9686 9244 8866 Maltitol 51980 51538 50142 49346 52760 50128 49178 51456 42808 51288 53814 44736 Mannitol 31282 32136 37278 35278 33060 32828 32118 33584 29096 30412 34110 28494 Methyl propenyl ketone 2198 1348 1914 1518 1816 1918 1382 1376 1602 1388 804 788 Methylhippuric acid 10346 8960 11408 9006 9244 8366 9436 9216 9998 9852 9046 8326 Metoprolol 10108 9642 11588 8048 5440 5228 7564 6978 9978 8684 12144 4282 Mevalonic acid 12134 12936 8652 12798 13394 13932 9600 10614 8826 12706 15198 8518 N,N-Dimethylformamide 1784 1778 1256 1438 1860 1662 1320 1480 1314 1476 1336 1640 N-Acetyl-L-alanine 18794 17904 18478 18492 19842 20254 18404 18370 16040 18316 19192 18888 N-Acetylglutamic acid 1342 1536 1898 1618 2032 1992 1246 1338 2434 1602 1844 1968 N-Acetylserotonin 9384 7554 14038 14674 7716 7318 6500 7008 9586 8794 8242 7996 N-Methyl-D-aspartic acid 3606 3110 3494 2706 2948 3366 2944 3050 4058 3682 4616 4010 Niacinamide 22670 28494 17816 16632 29940 32402 39296 37398 29988 28680 38984 38208 Nicotinic acid 34638 35708 36758 38134 43094 38720 37860 39480 29344 29164 36204 37874 Nutriacholic acid 218848 225166 142766 218216 216752 228326 177572 181198 130424 145980 193664 202292 o-Tyrosine 2696 2970 3736 4318 2476 1898 6838 7218 2334 2050 3430 3664 Pantothenic acid 50684 54620 48638 47322 52928 57644 50266 52986 60034 62082 60322 53504 Phenol 1124 1496 1380 1464 1414 1242 1594 1358 1582 1700 1492 1122 Phenylacetaldehyde 24748 22648 25432 22262 25380 23866 24410 25158 22346 20974 20490 22370 Phosphocreatine 77598 68144 122154 89846 59326 62064 62470 62458 96974 100496 137854 54804 Pregnenolone sulfate 58306 57046 55396 57772 58246 54498 44656 49996 50260 52320 51694 42458 Propionic acid 21260 16610 31718 22880 20650 22466 17378 16952 26378 20646 35860 24330 Propranolol 664 860 1212 998 1370 1052 1612 1326 642 1094 1060 788 Pyridine 5052 6520 4536 5480 5168 6522 8142 5434 7670 6258 5498 5948 Pyridoxine 2370 3306 2706 2634 2374 2482 4212 4930 3320 3574 2648 3430 Pyrrolidonecarboxylic acid 36384 34574 24684 27936 20614 20794 51648 56576 34494 37614 30326 32948 Pyruvaldehyde 11552 9632 10210 11064 10632 9154 7770 7854 9306 9080 9954 10956 Quinaldic acid 852 600 1730 1702 1922 1926 1466 1484 1890 2228 1806 1654 S-Adenosylhomocysteine 5020 5130 5084 5422 4538 5052 3452 3358 3104 3840 4192 3440 Saccharopine 6068 5022 8596 5414 4450 3254 3880 3004 8098 7112 9610 9802 Salicin 7530 7280 7458 6924 6610 7290 2248 1912 6284 1742 5468 4286 Sebacic acid 34042 28706 35150 41002 31508 30664 19612 24716 36758 33300 39314 38276 Spermine 42508 44976 46800 45118 41352 43814 40898 46682 42638 41696 40994 41436 Sphinganine 15082 15686 14536 13746 13150 9282 4650 4882 2920 2496 3836 3154 Sphingosine 11128 11422 11436 11198 13060 11472 8472 8234 5922 5126 7252 6254 Succinylacetone 18272 17694 9496 17250 17952 16470 11660 13046 7994 9126 16048 15328 Terephthalic acid 5604 4500 5690 5274 3096 2422 1732 2386 4974 3368 5580 1956 Trimethylamine 5110 4944 5088 3504 4444 4230 4292 5128 5216 4592 5866 5610 Uracil 6152 6462 6628 6874 7412 6954 5950 6392 5946 6928 6328 4900 Urea 3684 2966 2682 3084 2266 2668 3074 2148 2300 2570 2520 2424 Uric acid 19608 21062 23310 23642 23974 26662 26702 28906 30828 29178 28750 30910 Uridine 12028 10122 7492 9124 9802 9498 6568 6938 3350 2912 7088 4808 Xanthine 2252 2306 2998 2626 3654 3018 1522 1346 2578 1780 2064 1926 Xanthosine 8336 6464 6430 6428 8276 7240 3764 3770 4274 4942 3326 3122 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Molecular Formula RT [min] m/z meas. M meas. Ions MS/MS 1-Methyladenosine C11H15N5O4 2.21 282.11974 281.11247 [M+H]+ TRUE 17-Hydroxyprogesterone C21H30O3 19.45 331.22475 330.21748 [M+H]+ TRUE 2'-Deoxyguanosine 5'-monophosphate C10H14N5O7P 5.35 348.0704 347.06312 [M+H]+ TRUE 2,4-Diaminobutyric acid C4H10N2O2 13.6 119.08565 118.07837 [M+H]+ TRUE 2-Ethyl-2-hydroxybutyric acid C6H12O3 6.18 133.08591 132.07863 [M+H]+ TRUE 2-Hydroxyadenine C5H5N5O 4.75 152.05672 151.04945 [M+H]+ TRUE 2-Phenylaminoadenosine C16H18N6O4 10.96 381.13087 358.14169 [M+Na]+, [M+H]+, [M+K]+ TRUE 2-Phenylbutyric acid C10H12O2 15.6 165.09109 164.08382 [M+H]+ TRUE 2-Pyrrolidinone C4H7NO 6.76 86.06022 85.05295 [M+H]+ TRUE 3,4-Dihydroxyphenylglycol C8H10O4 2.41 171.06284 170.05556 [M+H]+ TRUE 3-Hydroxy-4-methoxycinnamic aicd C10H10O4 5.52 195.06285 194.05557 [M+H]+ TRUE 3-Methylindole C9H9N 5.97 132.08073 131.07346 [M+H]+ TRUE 3a,6b,7b-Trihydroxy-5b-cholanoic acid C24H40O5 7.42 409.29251 408.28524 [M+H]+ TRUE 5'-Methylthioadenosine C11H15N5O3S 5.81 298.09682 297.08954 [M+H]+ TRUE 5-Aminopentanoic acid C5H11NO2 1.6 118.08619 117.07892 [M+H]+ TRUE 5-Methoxysalicylic acid C8H8O4 10.23 169.04308 168.0358 [M+H]+ TRUE 5-Methoxytryptophol C11H13NO2 9.67 192.1022 191.09493 [M+H]+ TRUE 7,8-Dihydrobiopterin C9H13N5O3 1.65 240.10921 239.10194 [M+H]+ TRUE Acetoin C4H8O2 8.01 89.05973 88.05246 [M+H]+ TRUE Adenine C5H5N5 4.6 136.06172 135.05444 [M+H]+ TRUE Adenosine C10H13N5O4 4.6 268.10398 267.09671 [M+H]+ TRUE Adenosine 3',5'-diphosphate C10H15N5O10P2 2.18 428.03673 427.02946 [M+H]+ TRUE Adenosine monophosphate C10H14N5O7P 2.13 348.07039 347.06311 [M+H]+ TRUE Adenosine triphosphate C10H16N5O13P3 2.11 508.00305 506.99577 [M+H]+ TRUE Allocholic acid C24H40O5 13.38 409.2952 408.28792 [M+H]+ TRUE Androstenedione C19H26O2 9.16 287.19684 286.18993 [M+H]+, [M+K]+ TRUE Aspartame C14H18N2O5 5.27 295.1276 294.12033 [M+H]+ TRUE Aspirin C9H8O4 4.16 181.0472 180.03992 [M+H]+ TRUE Atenolol C14H22N2O3 2.12 267.17036 266.16308 [M+H]+ TRUE Benzoic acid C7H6O2 3.26 123.04403 122.03675 [M+H]+ TRUE Carnosine C9H14N4O3 1.69 249.09452 226.10532 [M+Na]+, [M+K]+ TRUE Cinnamaldehyde C9H8O 13.27 133.06484 132.05757 [M+H]+, [M-H2O+H]+ TRUE Clotrimazole C22H17ClN2 1.62 345.10913 344.10185 [M+H]+ TRUE Coumarin C9H6O2 3.27 147.04411 146.03683 [M+H]+ TRUE Cyclic AMP C10H12N5O6P 2.16 330.06004 329.05276 [M+H]+ TRUE Cytosine C4H5N3O 3.59 112.05042 111.04315 [M+H]+ TRUE D-Glucurono-6,3-lactone C6H8O6 3.78 177.03289 176.02561 [M+H]+ TRUE Diacetyl C4H6O2 8.59 87.04422 86.03694 [M+H]+ TRUE Diethanolamine C4H11NO2 10.23 106.08627 105.079 [M+H]+ TRUE Dopamine C8H11NO2 5.55 154.08632 153.07904 [M+H]+ TRUE Elaidic acid C18H34O2 17.94 283.26337 282.2561 [M+H]+, [M-H2O+H]+ TRUE Epinephrine C9H13NO3 5.51 184.09804 183.09076 [M+H]+ TRUE Folic acid C19H19N7O6 6.15 442.14723 441.13996 [M+H]+ TRUE Galactitol C6H14O6 1.93 183.08636 182.07908 [M+H]+ TRUE Gamma-Caprolactone C6H10O2 7.71 58.04142 114.06829 [M+H+H]2+ TRUE Gluconolactone C6H10O6 2.14 179.04849 178.04121 [M+H]+ TRUE Glucosamine C6H13NO5 5.46 180.08797 179.0807 [M+H]+ TRUE Glutathione C10H17N3O6S 2.15 308.09106 307.08333 [M+H]+, [M+Na]+, [M-H2O+H]+ TRUE Glycerophosphocholine C8H20NO6P 1.15 258.11012 257.10284 [M+H]+ TRUE Guanidinosuccinic acid C5H9N3O4 10.15 176.07068 175.06345 [M+H]+, [M+Na]+ TRUE Guanosine C10H13N5O5 4.75 284.09898 283.0917 [M+H]+ TRUE Guanosine 5'-diphosphate C10H15N5O11P2 1.77 444.03172 443.02444 [M+H]+ TRUE Guanosine monophosphate C10H14N5O8P 3.78 364.06333 363.05606 [M+H]+ TRUE Hydroxyisocaproic acid C6H12O3 14.5 133.08603 132.07876 [M+H]+ TRUE Hyodeoxycholic acid C24H40O4 20.16 393.29791 392.29063 [M+H]+ TRUE Hypoxanthine C5H4N4O 4.76 137.04572 136.03845 [M+H]+, [M-H2O+H]+ TRUE Indole C8H7N 5.97 118.06501 117.05774 [M+H]+ TRUE Indoleacrylic acid C11H9NO2 5.97 188.07066 187.06339 [M+H]+, [M-H2O+H]+ TRUE Inosine C10H12N4O5 4.76 269.08814 268.0811 [M+H]+, [M+K]+, [M+Na]+ TRUE Isobutyric acid C4H8O2 7.93 89.05975 88.05247 [M+H]+ TRUE Isovalerylcarnitine C12H23NO4 6.34 246.17005 245.16278 [M+H]+ TRUE L-Acetylcarnitine C9H17NO4 1.84 204.12308 203.1158 [M+H]+ TRUE L-Fucose C6H12O5 1.24 165.07583 164.06856 [M+H]+ TRUE L-Glutamic acid C5H9NO4 5.03 148.06058 147.0533 [M+H]+ TRUE L-Leucine C6H13NO2 3.26 132.10191 131.09463 [M+H]+ TRUE L-Methionine C5H11NO2S 2 150.05829 149.05101 [M+H]+ TRUE L-Norleucine C6H13NO2 9.16 132.10198 131.0947 [M+H]+ TRUE L-Phenylalanine C9H11NO2 5.13 166.08634 165.07907 [M+H]+ TRUE L-Proline C5H9NO2 1.27 116.07058 115.0633 [M+H]+ TRUE L-Thyronine C15H15NO4 3.78 274.10296 273.09568 [M+H]+ TRUE L-Tryptophan C11H12N2O2 5.97 205.09726 204.08998 [M+H]+ TRUE L-Tyrosine C9H11NO3 4.84 182.08122 181.07394 [M+H]+ TRUE Leucinic acid C6H12O3 6.46 133.0859 132.07862 [M+H]+ TRUE m-Coumaric acid C9H8O3 3.27 165.0547 164.04742 [M+H]+ TRUE Maltitol C12H24O11 1.23 345.13916 344.13188 [M+H]+ TRUE Mannitol C6H14O6 1.24 183.08635 182.07907 [M+H]+ TRUE Methyl propenyl ketone C5H8O 6.33 85.06502 84.05774 [M+H]+ TRUE Methylhippuric acid C10H11NO3 5.54 194.07884 193.07156 [M+H]+ TRUE Metoprolol C15H25NO3 11.51 268.18841 267.18114 [M+H]+ TRUE Mevalonic acid C6H12O4 4.41 171.06285 148.07356 [M+Na]+, [M+H]+, [M-H2O+H]+ TRUE N,N-Dimethylformamide C3H7NO 1.34 74.06019 73.05292 [M+H]+ TRUE N-Acetyl-L-alanine C5H9NO3 1.33 132.0655 131.0582 [M+H]+, [M-H2O+H]+ TRUE N-Acetylglutamic acid C7H11NO5 2.89 212.05301 189.06381 [M+Na]+, [M+H]+ TRUE N-Acetylserotonin C12H14N2O2 6.05 219.1105 218.10322 [M+H]+ TRUE N-Methyl-D-aspartic acid C5H9NO4 1.2 148.06055 147.05327 [M+H]+ TRUE Niacinamide C6H6N2O 2.23 123.05522 122.04794 [M+H]+ TRUE Nicotinic acid C6H5NO2 2.17 124.03923 123.03196 [M+H]+ TRUE Nutriacholic acid C24H38O4 20.08 413.26653 390.27739 [M+Na]+, [M+H]+, [M+K]+ TRUE o-Tyrosine C9H11NO3 5.34 182.08127 181.074 [M+H]+ TRUE Pantothenic acid C9H17NO5 5.51 220.11816 219.11072 [M+H]+, [M+Na]+, [M-H2O+H]+ TRUE Phenol C6H6O 3.25 95.04913 94.04186 [M+H]+ TRUE Phenylacetaldehyde C8H8O 16.56 121.06496 120.05768 [M+H]+ TRUE Phosphocreatine C4H10N3O5P 12.14 212.03759 211.03032 [M+H]+ TRUE Pregnenolone sulfate C21H32O5S 13.26 397.20114 396.19386 [M+H]+ TRUE Propionic acid C3H6O2 8.83 75.04413 74.03686 [M+H]+, [M-H2O+H]+ TRUE Propranolol C16H21NO2 5.25 260.16042 259.15314 [M+H]+ TRUE Pyridine C5H5N 2.17 80.04965 79.04237 [M+H]+ TRUE Pyridoxine C8H11NO3 2.28 152.07065 169.07391 [M-H2O+H]+, [M+H]+ TRUE Pyrrolidonecarboxylic acid C5H7NO3 2.38 130.04984 129.04258 [M+H]+, [M+Na]+ TRUE Pyruvaldehyde C3H4O2 7.73 73.02844 72.02117 [M+H]+ TRUE Quinaldic acid C10H7NO2 2.28 174.05837 173.05109 [M+H]+ TRUE S-Adenosylhomocysteine C14H20N6O5S 4.11 385.12895 384.12167 [M+H]+ TRUE Saccharopine C11H20N2O6 11.31 277.14116 276.13388 [M+H]+ TRUE Salicin C13H18O7 5.31 287.1064 286.09912 [M+H]+ TRUE Sebacic acid C10H18O4 8.57 102.06772 202.12089 [M+H+H]2+ TRUE Spermine C10H26N4 0.79 203.22309 202.21581 [M+H]+ TRUE Sphinganine C18H39NO2 11.29 302.30547 301.29819 [M+H]+ TRUE Sphingosine C18H37NO2 13.13 300.28989 299.28262 [M+H]+ TRUE Succinylacetone C7H10O4 18.84 159.06523 158.05796 [M+H]+ TRUE Terephthalic acid C8H6O4 20.08 149.02343 166.02672 [M-H2O+H]+, [M+H]+ TRUE Trimethylamine C3H9N 1.85 60.08089 59.07361 [M+H]+ TRUE Uracil C4H4N2O2 3.4 113.03448 112.02721 [M+H]+ TRUE Urea CH4N2O 21.6 61.03974 60.03247 [M+H]+ TRUE Uric acid C5H4N4O3 2.5 169.03185 168.02457 [M+H]+ TRUE Uridine C9H12N2O6 3.41 245.07684 244.06956 [M+H]+ TRUE Xanthine C5H4N4O2 5.01 153.04078 152.03351 [M+H]+ TRUE Xanthosine C10H12N4O6 5.01 285.08313 284.07586 [M+H]+ TRUE METABOLITES_END #END