#METABOLOMICS WORKBENCH SIMR_Core_Facility_20230607_023253 DATATRACK_ID:4068 STUDY_ID:ST002731 ANALYSIS_ID:AN004428 PROJECT_ID:PR001695 VERSION 1 CREATED_ON June 7, 2023, 5:48 am #PROJECT PR:PROJECT_TITLE Untargeted Multi-Omics of LNCaP Cell-line Treated with Novel DNA Minor Groove PR:PROJECT_TITLE Binder and /or Doxorubicin Using Mass-Spectrometry PR:PROJECT_SUMMARY Prostate cancer poses a significant health risk, ranking as the second most PR:PROJECT_SUMMARY common cancer among men in the United States. However, the effectiveness of PR:PROJECT_SUMMARY current anti-prostate cancer drugs is limited due to increasing drug resistance PR:PROJECT_SUMMARY and side effects. Consequently, there is a pressing need to develop new PR:PROJECT_SUMMARY compounds and identify novel drug targets that can surpass these limitations. PR:PROJECT_SUMMARY Due to their targeted mechanism, DNA minor groove binders (MGBs) are becoming PR:PROJECT_SUMMARY more popular as a relatively safe and effective alternative. In our research, we PR:PROJECT_SUMMARY employed multi-omics techniques to investigate the mechanism of action of a PR:PROJECT_SUMMARY novel MGB compound (MGB4) through LC-MS/MS-based untargeted metabolomics PR:PROJECT_SUMMARY combined with discovery proteomics analysis performed on LNCaP cells, which were PR:PROJECT_SUMMARY treated with MGB4, doxorubicin, or a combination of both compounds. Through a PR:PROJECT_SUMMARY one-way ANOVA test with a significance level of p-value < 0.05, we identified 99 PR:PROJECT_SUMMARY metabolites and 1143 proteins associated with the treatments. Our findings PR:PROJECT_SUMMARY indicate that treating LNCaP cells with doxorubicin or the MGB4 lead compound PR:PROJECT_SUMMARY yielded similar effects, albeit not identical, on the cells. Both compounds PR:PROJECT_SUMMARY deactivated the translation pathway in the cells. Furthermore, we observed PR:PROJECT_SUMMARY alterations in sphingolipid and amino acid metabolic pathways, potentially PR:PROJECT_SUMMARY contributing to the suppression of prostate cancer cell proliferation and PR:PROJECT_SUMMARY division. Additionally, doxorubicin and combined treatments resulted in reduced PR:PROJECT_SUMMARY metabolism of spermine and spermidine, likely stemming from decreased protein PR:PROJECT_SUMMARY synthesis of key enzymes involved in their pathways. Moreover, the combined PR:PROJECT_SUMMARY treatment exhibited a synergistic interaction between the two compounds, leading PR:PROJECT_SUMMARY to altered purine metabolism and a more pronounced reduction in metabolite PR:PROJECT_SUMMARY abundance compared to individual treatments. Overall, our study demonstrates the PR:PROJECT_SUMMARY robustness of the multi-omics approach in elucidating the mechanism of action of PR:PROJECT_SUMMARY promising drug candidates. It also suggests that MGB4 shows potential as a PR:PROJECT_SUMMARY candidate for prostate cancer treatment. 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, Sharjah, PR:ADDRESS UAE, Sharjah, 000, United Arab Emirates PR:EMAIL tims-tof@sharjah.ac.ae PR:PHONE +971 6 5057656 #STUDY ST:STUDY_TITLE Untargeted Multi-Omics of LNCaP Cell-line Treated with Novel DNA Minor Groove ST:STUDY_TITLE Binder and /or Doxorubicin Using Mass-Spectrometry ST:STUDY_SUMMARY Prostate cancer poses a significant health risk, ranking as the second most ST:STUDY_SUMMARY common cancer among men in the United States. However, the effectiveness of ST:STUDY_SUMMARY current anti-prostate cancer drugs is limited due to increasing drug resistance ST:STUDY_SUMMARY and side effects. Consequently, there is a pressing need to develop new ST:STUDY_SUMMARY compounds and identify novel drug targets that can surpass these limitations. ST:STUDY_SUMMARY Due to their targeted mechanism, DNA minor groove binders (MGBs) are becoming ST:STUDY_SUMMARY more popular as a relatively safe and effective alternative. In our research, we ST:STUDY_SUMMARY employed multi-omics techniques to investigate the mechanism of action of a ST:STUDY_SUMMARY novel MGB compound (MGB4) through LC-MS/MS-based untargeted metabolomics ST:STUDY_SUMMARY combined with discovery proteomics analysis performed on LNCaP cells, which were ST:STUDY_SUMMARY treated with MGB4, doxorubicin, or a combination of both compounds. Through a ST:STUDY_SUMMARY one-way ANOVA test with a significance level of p-value < 0.05, we identified 99 ST:STUDY_SUMMARY metabolites and 1143 proteins associated with the treatments. Our findings ST:STUDY_SUMMARY indicate that treating LNCaP cells with doxorubicin or the MGB4 lead compound ST:STUDY_SUMMARY yielded similar effects, albeit not identical, on the cells. Both compounds ST:STUDY_SUMMARY deactivated the translation pathway in the cells. Furthermore, we observed ST:STUDY_SUMMARY alterations in sphingolipid and amino acid metabolic pathways, potentially ST:STUDY_SUMMARY contributing to the suppression of prostate cancer cell proliferation and ST:STUDY_SUMMARY division. Additionally, doxorubicin and combined treatments resulted in reduced ST:STUDY_SUMMARY metabolism of spermine and spermidine, likely stemming from decreased protein ST:STUDY_SUMMARY synthesis of key enzymes involved in their pathways. Moreover, the combined ST:STUDY_SUMMARY treatment exhibited a synergistic interaction between the two compounds, leading ST:STUDY_SUMMARY to altered purine metabolism and a more pronounced reduction in metabolite ST:STUDY_SUMMARY abundance compared to individual treatments. Overall, our study demonstrates the ST:STUDY_SUMMARY robustness of the multi-omics approach in elucidating the mechanism of action of ST:STUDY_SUMMARY promising drug candidates. It also suggests that MGB4 shows potential as a ST:STUDY_SUMMARY candidate for prostate cancer treatment. 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 Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male #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 - 1-01-8388 Group:Control RAW_FILE_NAME=1-01-8388.d SUBJECT_SAMPLE_FACTORS - 1-02-8389 Group:Control RAW_FILE_NAME=1-02-8389.d SUBJECT_SAMPLE_FACTORS - 1'-01-8390 Group:Control RAW_FILE_NAME=1'-01-8390.d SUBJECT_SAMPLE_FACTORS - 1'-02-8391 Group:Control RAW_FILE_NAME=1'-02-8391.d SUBJECT_SAMPLE_FACTORS - 1''-01-8392 Group:Control RAW_FILE_NAME=1''-01-8392.d SUBJECT_SAMPLE_FACTORS - 1''-02-8393 Group:Control RAW_FILE_NAME=1''-02-8393.d SUBJECT_SAMPLE_FACTORS - 2-01-8394 Group:MGB4 RAW_FILE_NAME=2-01-8394.d SUBJECT_SAMPLE_FACTORS - 2-02-8395 Group:MGB4 RAW_FILE_NAME=2-02-8395.d SUBJECT_SAMPLE_FACTORS - 2'-01-8396 Group:MGB4 RAW_FILE_NAME=2'-01-8396.d SUBJECT_SAMPLE_FACTORS - 2'-02-8397 Group:MGB4 RAW_FILE_NAME=2'-02-8397.d SUBJECT_SAMPLE_FACTORS - 2''-01-8398 Group:MGB4 RAW_FILE_NAME=2''-01-8398.d SUBJECT_SAMPLE_FACTORS - 2''-02-8399 Group:MGB4 RAW_FILE_NAME=2''-02-8399.d SUBJECT_SAMPLE_FACTORS - 3-01-8401 Group:Dox RAW_FILE_NAME=3-01-8401.d SUBJECT_SAMPLE_FACTORS - 3-02-8402 Group:Dox RAW_FILE_NAME=3-02-8402.d SUBJECT_SAMPLE_FACTORS - 3'-01-8403 Group:Dox RAW_FILE_NAME=3'-01-8403.d SUBJECT_SAMPLE_FACTORS - 3'-02-8404 Group:Dox RAW_FILE_NAME=3'-02-8404.d SUBJECT_SAMPLE_FACTORS - 3''-01-8405 Group:Dox RAW_FILE_NAME=3''-01-8405.d SUBJECT_SAMPLE_FACTORS - 3''-02-8406 Group:Dox RAW_FILE_NAME=3''-02-8406.d SUBJECT_SAMPLE_FACTORS - 4-01-8407 Group:Combined RAW_FILE_NAME=4-01-8407.d SUBJECT_SAMPLE_FACTORS - 4-02-8408 Group:Combined RAW_FILE_NAME=4-02-8408.d SUBJECT_SAMPLE_FACTORS - 4'-01-8409 Group:Combined RAW_FILE_NAME=4'-01-8409.d SUBJECT_SAMPLE_FACTORS - 4'-02-8410 Group:Combined RAW_FILE_NAME=4'-02-8410.d SUBJECT_SAMPLE_FACTORS - 4''-01-8411 Group:Combined RAW_FILE_NAME=4''-01-8411.d SUBJECT_SAMPLE_FACTORS - 4''-02-8412 Group:Combined RAW_FILE_NAME=4''-02-8412.d #COLLECTION CO:COLLECTION_SUMMARY Human prostate cancer cells LNCaP were grown in Roswell Park Memorial Institute CO:COLLECTION_SUMMARY (RPMI) 1640 Medium (Sigma Aldrich, Germany) supplemented with 10% fetal bovine CO:COLLECTION_SUMMARY serum (FBS) (Sigma Aldrich, Germany). A combination of 1 % penicillin and CO:COLLECTION_SUMMARY streptomycin antibiotics was added to RPMI/FBS growth media. Cells were CO:COLLECTION_SUMMARY maintained in humidified incubator with conditions of 37◦C temperature and 5 % CO:COLLECTION_SUMMARY CO2. CO:SAMPLE_TYPE LNCaP cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Three biological replicates were prepared for each treatment condition in T75 TR:TREATMENT_SUMMARY cm2 flasks (MGB4 and/or Doxorubicin and control group), and each replicate was TR:TREATMENT_SUMMARY seeded with approximately 2 × 106 LNCaP cells, then separately treated with TR:TREATMENT_SUMMARY MGB4 (2.15 μM) and/or Doxorubicin (1.18 μM) and incubated for 24 h. A uniform TR:TREATMENT_SUMMARY number of cells was seeded in T75 cm2 flask for each sample to avoid the effect TR:TREATMENT_SUMMARY of variable cell number on the outcome of the experiment. After incubation, TR:TREATMENT_SUMMARY cells were washed twice with phosphate-buffered saline solution (PBS) and then TR:TREATMENT_SUMMARY collected by trypsinization. Finally, cells were collected as pellets by TR:TREATMENT_SUMMARY centrifugation at 1200 rounds per minute (rpm) for 10 min at room temperature TR:TREATMENT_SUMMARY and resuspended in 1 mL 1× PBS for further analysis. Cells were maintained in TR:TREATMENT_SUMMARY the same conditions during incubation, and all samples were collected TR:TREATMENT_SUMMARY simultaneously (Figure 5). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY we used chloroform/methanol extraction protocol to increase the coverage of the SP:SAMPLEPREP_SUMMARY extracted metabolites. At first, the samples (cells and buffer) were transferred SP:SAMPLEPREP_SUMMARY into Eppendorf tubes then centrifuged at 14000 rpm for 5 min. Afterward, the SP:SAMPLEPREP_SUMMARY buffer was discarded, and the cells were preserved. To each sample, 400 µL of SP:SAMPLEPREP_SUMMARY the mixture containing one protease inhibitor tablet and 10 mL of lysis buffer SP:SAMPLEPREP_SUMMARY was added. Following rest for 10 minutes, samples were transferred to 10 mL SP:SAMPLEPREP_SUMMARY tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator SP:SAMPLEPREP_SUMMARY (QSONICA SONICATOR, USA) for 30 seconds while utilizing a 30 % amplifier in an SP:SAMPLEPREP_SUMMARY ice bath. The samples were then transferred to Eppendorf tubes and centrifuged SP:SAMPLEPREP_SUMMARY for 5 minutes at 14000 rpm. The supernatant was then transferred to another SP:SAMPLEPREP_SUMMARY Eppendorf, and 400 µL of methanol and 300 µL of chloroform were added. SP:SAMPLEPREP_SUMMARY Following that, the samples were vortexed for 30 seconds and centrifuged for 5 SP:SAMPLEPREP_SUMMARY minutes at 14000 rpm. After that, two metabolite-containing layers are obtained, SP:SAMPLEPREP_SUMMARY after transferring the upper layer of each sample to glass vials, 400 µl of SP:SAMPLEPREP_SUMMARY methanol was added, followed by vertexing and centrifugation. The remaining SP:SAMPLEPREP_SUMMARY supernatant was transferred to the same glass vials used before for the drying SP:SAMPLEPREP_SUMMARY step, with the remaining protein pellets being air-dried for proteomics. A dried SP:SAMPLEPREP_SUMMARY metabolomics sample was resuspended in 200 µL (0.1% formic acid in water) and SP:SAMPLEPREP_SUMMARY injected into HPLC to be analysed by Q-TOF MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were chromatographically separated by inline reversed-phase CH:CHROMATOGRAPHY_SUMMARY chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, CH:CHROMATOGRAPHY_SUMMARY Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B CH:CHROMATOGRAPHY_SUMMARY 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm x 2.1 mm, 1.8µm CH:CHROMATOGRAPHY_SUMMARY beads) was maintained at 35C. For metabolomics, 10 µL was injected twice for CH:CHROMATOGRAPHY_SUMMARY each sample and eluted using a 30-minute gradient as follows: 1% ACN was held CH:CHROMATOGRAPHY_SUMMARY for 2 minutes, ramping to 99% ACN over 15 minutes, held at 99% ACN for 3 minutes CH:CHROMATOGRAPHY_SUMMARY before re-equilibrating to 1% ACN for 10 minutes. Flow rates were 250 µL/min CH:CHROMATOGRAPHY_SUMMARY for elution and 350 µL/min for re-equilibration. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Bruker Elute CH:COLUMN_NAME Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 1%B to 99%B in 15 min CH:FLOW_RATE 250 uL/min CH:COLUMN_TEMPERATURE 35 #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 MS analysis was performed using a TimsTOF (Bruker, Darmstadt, Germany) with MS:MS_COMMENTS Apollo II electrospray ionization (ESI) source. The drying gas was set to flow MS:MS_COMMENTS at 10 L/min and the drying temperature to 220C and the nebulizer pressure to 2.2 MS:MS_COMMENTS bar. The capillary voltage was 4500 V and the end plate offset 500V. For MS:MS_COMMENTS metabolomics the scan range was 20-1300 m/z. The collision energy was set to 20 MS:MS_COMMENTS eV, the cycle time to 0.5 seconds with a relative minimum intensity threshold of MS:MS_COMMENTS 400 counts per thousand and target intensity of 20,000. Sodium formate was MS:MS_COMMENTS injected as an external calibrant in the first 0.3 minutes of each LC-MS/MS run. MS:MS_COMMENTS MetaboScape 4.0 software was used for metabolite processing and statistical MS:MS_COMMENTS analysis (Bruker Daltonics). The following parameters for molecular feature MS:MS_COMMENTS identification and "bucketing" were set in the T-ReX 2D/3D workflow: For peak MS:MS_COMMENTS detection, a minimum intensity threshold of 1,000 counts is required, as well as MS:MS_COMMENTS a minimum peak duration of 7 spectra, with feature quantification determine MS:MS_COMMENTS using peak area. The file masses were recalibrated based on the external MS:MS_COMMENTS calibrant injected between 0-0.3 min. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples 1-01-8388 1-02-8389 1'-01-8390 1'-02-8391 1''-01-8392 1''-02-8393 2-01-8394 2-02-8395 2'-01-8396 2'-02-8397 2''-01-8398 2''-02-8399 3-01-8401 3-02-8402 3'-01-8403 3'-02-8404 3''-01-8405 3''-02-8406 4-01-8407 4-02-8408 4'-01-8409 4'-02-8410 4''-01-8411 4''-02-8412 Factors Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Group:MGB4 Group:MGB4 Group:MGB4 Group:MGB4 Group:MGB4 Group:MGB4 Group:Dox Group:Dox Group:Dox Group:Dox Group:Dox Group:Dox Group:Combined Group:Combined Group:Combined Group:Combined Group:Combined Group:Combined 17-Hydroxyprogesterone 11928 15680 11986 13046 13306 13450 11948 10086 11160 12160 14604 11676 9376 10308 9164 9242 12882 11512 10358 9410 11650 8702 9188 10370 2'-Deoxyguanosine 5'-monophosphate 5062 8472 7512 7978 6192 6048 8412 9746 10728 11382 8862 8794 7580 7206 9736 8392 11200 11612 4214 4094 5102 4442 4518 5262 2-Ethyl-2-hydroxybutyric acid 14434 14866 15192 19052 15242 15304 11818 12726 12954 12014 13872 13204 13884 12810 12916 14150 14376 15472 11404 11200 11306 11178 12248 11064 2-Hydroxyadenine 11898 8132 11258 10888 14252 10218 5672 6652 7774 8412 7040 7654 7432 9066 6782 8830 8342 9814 6428 5718 6320 6034 5658 5936 2-Phenylaminoadenosine 55544 59318 34492 30258 33770 36528 13972 17026 24130 24008 19290 21856 24540 25836 22334 22410 20712 24588 27992 31016 12046 11792 25428 26374 2-Phenylbutyric acid 22384 23344 19840 19804 14394 18706 8376 7580 9086 8864 8520 8954 10012 11626 8430 10408 8822 9328 17988 16434 7844 8928 17266 15830 2-Pyrrolidinone 5952 5512 4946 5670 5198 5420 4820 4704 4874 4600 4542 5270 4796 5136 4644 4526 5330 5010 3512 3424 3870 4986 4290 3710 3,4-Dihydroxyphenylglycol 11272 43596 13254 8650 40116 19270 28362 50800 16458 11748 18792 17108 2386 2418 24040 2584 17238 13156 16402 8832 22170 35886 1822 2372 3,5-Diiodo-L-tyrosine 404 506 748 872 838 668 3086 3182 3312 2864 994 3074 3288 3154 3470 5216 1960 2654 1264 894 0 0 2898 2330 3-Nitrotyrosine 1900 1920 2370 2282 2030 2038 908 1244 1774 1042 1436 1072 598 0 1056 886 882 730 0 0 0 414 558 0 3-Phenylbutyric acid 30310 28224 24706 24850 27504 24340 10408 9884 13178 10636 11306 12046 14172 15260 11108 11160 14480 13518 25052 20548 12276 12836 18998 21726 3a,6b,7b-Trihydroxy-5b-cholanoic acid 55086 63716 58970 55638 52728 54452 51792 54010 57376 53354 58326 58534 50010 54850 50878 45494 56076 54788 33526 35184 49008 49020 57430 53394 5'-Methylthioadenosine 102354 110272 91230 106318 126516 133938 96578 101508 116230 93584 114690 114310 39736 38572 50446 43002 48428 49724 29336 34666 28328 27352 25316 24848 5-Methoxytryptophol 17964 14462 11594 10868 6020 5804 14286 14776 10190 9334 6800 7642 18280 18396 13978 13250 16062 14102 11882 13288 12906 13784 6568 7414 7,8-Dihydrobiopterin 872 858 1094 1056 1164 1172 682 644 994 722 756 906 674 942 624 680 804 920 614 570 646 520 604 642 Acetoin 27172 33254 29876 29742 32448 28682 18850 19236 17458 22162 21888 22280 19222 17990 18744 19690 20360 20904 14368 12792 14534 14904 13830 13660 Acetone 1018 1410 1668 2030 1030 2358 2012 1784 2134 1948 1454 1506 1714 1748 1734 1408 1686 2012 2018 1576 1976 1788 1402 1746 Adenine 35506 35450 33228 37616 51938 48500 30554 34208 37756 34504 37546 39494 12202 12460 15240 13106 14250 13204 9842 7848 8456 8338 7402 7994 Adenosine 60024 59756 43234 43877 88640 99960 34352 33676 33386 33985 32551 32032 54420 53722 52040 52832 42218 42774 25498 23710 42286 45310 26790 28956 Adenosine 3',5'-diphosphate 11778 11638 13568 12522 29238 26238 30992 29450 19998 18812 11230 14728 44398 43680 50444 51722 24998 25654 20952 20786 26144 21336 23474 24298 Adenosine monophosphate 5776 6488 6724 6952 7456 6918 2748 2024 3248 3900 4334 4522 3480 3200 3138 3134 4754 4814 2320 1960 3086 2988 2156 2266 Adenosine triphosphate 3912 2952 4372 4284 2108 2598 2282 3462 2798 2570 2908 3260 2336 2146 1984 2614 2398 1846 1776 1468 1520 1522 1522 1468 ADP 4240 5138 4884 5588 5488 5108 3008 3074 3310 2918 3242 4162 2868 2392 3080 3488 4604 3794 1508 1160 1600 1374 1560 1280 Alpha-N-phenylacetyl-L-glutamine 2730 2700 3424 3104 4580 3226 1096 1134 1452 1156 1308 1070 748 1386 1662 1630 1592 1464 804 922 1088 940 778 922 Aminopterin 2588 2356 3054 2646 3278 3516 1184 1336 1358 1430 1288 1262 1210 1338 1382 1212 1486 1490 578 618 620 694 510 621 Androstenedione 7248 9914 8964 7864 8646 7982 7862 9440 7902 9668 7522 8794 7306 7074 8398 10892 9268 10314 3902 3272 3986 3852 2782 2132 Benzene 0 0 0 0 184 994 0 0 142 0 278 230 3892 2376 4920 3694 0 0 1566 1520 1638 1617 1412 978 Benzocaine 2188 1762 1474 1636 1244 1716 1164 1286 1606 1386 1388 1442 1422 1454 1854 1714 1604 1962 1096 1322 1186 1018 1264 1176 Beta-Alanine 5364 7892 6572 7764 7626 7686 5646 4458 6480 5970 6950 7428 3354 3276 4926 3860 4758 3952 2378 1852 3158 2088 1418 1954 Carnosine 16846 18772 21658 19082 20574 17080 15558 20934 25808 18106 21404 22968 15008 16124 17928 17866 18434 19682 8384 10750 13764 15412 10062 10758 Chalcone 1422 1082 1522 1238 1442 2038 1294 1012 1500 1080 1318 1482 836 905 1224 1144 1348 1576 870 887 218 924 648 615 Cytosine 790 1182 1236 1242 1592 1254 1782 1808 1202 814 1420 1452 1038 1302 1144 1010 1106 608 984 702 1056 1044 698 344 D-Glucurono-6,3-lactone 1538 1298 2060 1390 2178 2354 864 624 776 590 1126 702 830 1062 1198 828 1221 1136 715 688 774 874 822 804 Diacetyl 6722 8696 8000 7912 6256 9054 4900 5294 6506 5698 5540 4710 4258 4196 4380 4316 5720 5160 2250 2398 3194 3390 2912 2730 Diethanolamine 25901 24688 12838 11938 11316 12284 4900 10856 10794 9898 10356 11966 14724 15648 15416 11398 14138 14920 715 20528 1028 1426 13412 9902 Ethanolamine 55800 53728 38150 42334 41020 45112 50810 51822 51378 39622 41972 48218 52000 65548 61612 50372 62332 58052 47150 47600 31360 30530 40264 43908 Ethyl malonate 4348 4132 3020 2874 3650 3310 2654 2816 2952 2866 2332 3178 3362 4144 4120 4006 3724 4244 1490 1562 2228 2016 2094 2402 Galactitol 30290 28646 30604 29654 26668 24398 29304 31488 27292 28004 28956 26850 32124 30224 26968 26858 29876 28728 36420 33852 31742 35346 34974 33682 Gamma-Glutamylcysteine 7060 9416 9238 8282 10596 10158 5432 6088 5856 6248 5902 7002 6816 5748 6756 5772 6204 6616 2434 2590 2782 4284 2064 2194 Gluconolactone 23340 29998 19118 17236 17462 26762 27662 31032 23794 21668 26784 25844 21124 22356 13942 29462 20526 18394 2210 4242 32278 29092 5776 4310 Glutathione 108946 124384 121035 122545 83658 108770 107750 121732 119274 105736 119008 123506 79752 85142 102420 102934 90325 88738 8288 9631 123788 117502 22218 18530 Glycerol 2046 1148 1904 1606 1438 1702 4150 5114 4202 3766 3310 4144 3782 3368 4424 4160 2964 3372 1248 1001 1356 1322 298 508 Glycerophosphocholine 4118 3228 3718 3480 3480 2726 5370 6120 6240 6448 6348 6698 4978 5082 3944 5032 5088 5386 7468 6790 7040 8212 5762 5494 Glycine 1510 1492 1238 776 962 1038 1218 906 770 708 1110 1284 762 646 840 734 908 1002 1238 874 272 334 694 772 Glycocholic acid 5818 3706 3642 3658 6476 8712 3236 2946 1742 1782 3786 3606 3382 4008 3404 3458 2230 3156 4032 4418 588 0 5000 5418 Glycylproline 4298 5310 4260 5230 2982 2066 4838 5128 5116 6634 4004 4454 6882 6126 5868 5302 4644 6268 5034 7582 5212 5926 3862 3610 Guanine 3420 4260 4798 3858 4398 3888 3062 3088 3610 3460 3380 4418 2862 2750 3688 3512 2996 3676 1126 1418 1622 1194 1700 1724 Guanosine 109080 157918 160148 85962 179622 168968 103878 108220 121468 103180 105914 116246 97866 99060 120464 114774 109108 108240 52490 55458 69770 68542 61420 53908 Guanosine 5'-diphosphate 3468 2696 6228 2504 8704 4842 1976 2186 3960 4100 4254 5002 3490 2262 2832 4212 3154 3298 2360 1498 3614 1912 3332 3468 Hydroxyisocaproic acid 22708 21208 19382 17098 17440 18432 7140 7606 8356 8488 8868 7800 10980 9708 8516 8702 9170 11814 15486 17724 8136 8592 13792 17144 Hyodeoxycholic acid 16426 17482 19554 23504 28130 14962 12412 24056 17660 10158 24188 26922 36408 35792 38070 39072 19982 30406 38730 30932 38296 36194 50900 57454 Hypoxanthine 321945 337706 347260 294316 351494 346518 251056 245482 267154 196880 246970 247900 206108 217432 227168 234066 245060 210050 133362 127590 147920 160690 117814 123060 Indole 14198 13760 13514 14564 12478 11560 8700 6692 8010 7328 6720 8160 5418 5866 6500 6144 6784 7538 2552 3156 5994 3476 2558 3184 Indole-3-carbinol 7720 6430 5258 5328 6368 4754 2882 3992 3314 3808 4426 3486 4462 4072 3908 3194 5120 4206 2616 3566 1918 1768 2680 2358 Indoleacrylic acid 79078 77932 77958 76692 62520 64726 45822 45376 51594 52558 49276 47410 36238 38516 44444 41260 51072 47216 23340 22904 19080 24306 20044 20616 Inosine 150760 153754 156768 155760 168028 173506 104778 102718 120146 118150 102982 116848 86902 90588 102992 104418 101290 100960 47562 41858 58318 60662 46642 42154 Isovalerylcarnitine 27192 27706 26368 21328 26462 30880 22176 20960 25440 25612 21416 25614 38416 37226 44568 43038 40886 44062 17300 16478 21920 26836 16788 15390 L-Acetylcarnitine 19060 19656 18216 19810 18722 17604 12064 10614 12636 16624 13180 13832 13008 13400 17750 15870 19728 25310 8316 5940 7556 7716 6060 5976 L-Dopa 1388 1980 2314 2116 2522 2040 1954 2596 3184 4054 2460 2224 1816 2304 2060 1516 1932 1806 2986 3406 4014 4018 3516 2966 L-Fucose 2084 2360 2046 1908 1868 1800 1958 2632 1914 1746 2832 1908 1798 2290 1882 2246 2032 2142 2698 2462 2064 2438 1762 2524 L-Homoserine 1452 990 956 792 1140 1136 1094 1278 736 754 1000 1082 1012 930 892 904 840 670 1180 1174 372 1485 698 1548 L-Norleucine 10226 10906 12142 12206 12264 13264 10470 11968 11206 11520 10494 11494 10454 10376 12548 10648 10878 12338 5024 5338 4568 5608 2326 3014 L-Phenylalanine 13416 15656 16082 13224 19544 21460 7692 7946 7836 7566 7084 8226 7720 7544 10026 8698 8400 8068 3106 3268 4952 3828 3586 3550 L-Tryptophan 14896 16628 15594 14838 12748 11788 6872 9156 9652 10788 9074 9224 6724 7754 7562 8356 8196 10538 4264 4522 5264 5152 3918 4166 Leucinic acid 16646 17000 21998 20478 22172 19160 13674 11894 14036 13840 15974 14326 13472 14692 13458 13002 16366 16398 14778 13958 15540 13518 12852 14636 Maltitol 52478 50184 56946 49596 53222 40938 47530 53084 45324 49430 48924 52898 54358 56334 45072 52146 46264 47966 53900 56732 53636 56552 59584 60372 Medroxyprogesterone 6060 3228 4354 6574 4552 4734 4388 4768 6182 5012 2592 6732 9536 7918 6994 6854 5214 6842 12456 8816 8047 9050 11002 9134 Melatonin 5212 4632 4882 4602 4850 4288 3038 3614 2504 3268 2462 3672 3160 3124 2208 2688 3742 2976 3605 3772 3918 4452 3958 3490 Methylmalonic acid 1310 1540 1826 2032 2386 2042 800 1426 1674 826 1146 1514 1344 1530 1244 1296 1086 1730 954 702 748 760 1152 1584 Mevalonic acid 10642 7178 7364 13818 8352 6088 5086 8790 13318 5254 7528 14912 5548 5584 4584 4840 9916 14248 3800 7578 3738 5482 4794 3002 N-Acetyl-L-alanine 19692 21488 18038 17332 16042 15843 17792 18486 16794 17702 16192 16580 18042 19062 18032 17834 16822 17694 16476 17264 16776 17034 16824 16266 N-Acetylserotonin 14426 14560 14980 15764 12430 11818 11770 12146 13538 13302 14108 12142 11432 12518 11938 11278 12694 12320 9936 12062 13170 13998 12102 13196 N-Methyl-a-aminoisobutyric acid 2704 2508 2180 2636 1982 1154 1204 1328 1494 2156 1458 1192 976 890 1504 1238 1388 2346 2284 874 814 618 1234 1718 N-Methyl-D-aspartic acid 5460 4702 6080 4902 6524 4814 2152 2590 2782 2670 3048 2742 2610 2996 2602 3004 3226 2956 2988 2214 3192 2822 2296 2260 Niacinamide 1504 1806 1756 1454 2258 2280 1426 1352 1714 630 914 1414 638 630 1054 788 1222 1002 164 294 420 714 310 315 Nutriacholic acid 186326 208394 214390 263158 232284 152478 131090 208370 153608 77964 194854 218892 324498 327114 293716 290738 217680 224192 359728 311056 182642 149346 376434 342104 Pantothenic acid 55540 58974 59484 57474 61692 65706 50788 51254 54412 56286 50892 57084 43744 38840 49840 41678 47618 45770 24016 26790 32986 35840 24928 25062 PC(16:0/16:0) 9332 7628 13492 9124 12590 8696 18512 17814 4954 5698 7992 7678 5094 3676 15282 10810 18124 16316 6280 4934 2924 2546 4112 3062 PC(18:1(9Z)/18:1(9Z)) 3536 4196 17654 19918 13314 15305 13684 11300 4028 13182 6810 4478 3238 1806 5604 6878 15374 16562 2286 1742 1468 5752 1036 1160 Phenylacetaldehyde 10462 9666 6310 7514 8836 6970 4252 3652 3548 3880 3080 3192 5020 4054 4744 2636 3922 4404 6774 5522 3864 3556 5682 6726 Phosphocreatine 161254 116286 81698 75100 79600 90018 20754 21838 0 25952 25176 31820 43150 45858 34704 32586 40904 43944 62102 69534 1216 0 37852 39902 Propionic acid 3888 3984 4152 6054 5046 4310 4038 5052 4288 4192 3954 4896 3996 4222 4116 4832 5484 4488 2528 1996 1366 1520 970 1080 Pyridine 3708 3944 3076 3002 4368 4866 4606 3164 1902 2464 1928 2026 2064 2662 2494 2016 2420 3472 1612 2530 5070 4134 1054 1214 Pyridoxal 1512 2576 1084 1190 1354 826 2632 2404 1660 1200 1528 1574 2482 2000 2496 2436 2566 1722 3116 3424 2888 3758 2950 2806 Pyridoxal 5'-phosphate 150 244 0 216 408 410 305 356 400 0 0 0 2542 2120 3050 3256 3006 2100 2004 1150 2190 1901 1626 1662 Pyrrolidonecarboxylic acid 30162 28434 34156 35580 42558 35112 9064 9776 11750 12906 13692 11228 15026 16292 11784 10868 17384 16850 20004 18508 6094 5904 10006 11190 Pyruvaldehyde 4896 4784 5740 4598 6206 4374 4182 4288 4488 4002 4036 4438 4856 4266 5144 4654 4642 3980 4428 4204 4110 4902 5358 4874 Salicin 3022 3206 2480 2406 2370 2648 1870 1334 1586 1608 1586 1922 1592 1450 1472 1224 2016 1592 726 530 840 968 682 594 Sebacic acid 27988 21560 18172 17404 20702 20458 7976 8498 10330 8366 8201 8110 11962 10178 8210 8272 11992 11094 20324 15496 10306 10820 11250 11222 Spermine 62532 62046 62008 59702 56156 53894 39414 43216 50546 42014 43328 45400 30706 29074 35934 38758 50730 44118 308 302 7568 7376 4364 4750 Sphinganine 9986 8670 5180 5082 7114 7858 984 1126 1680 1120 1340 1148 2388 0 1582 1518 1636 1868 2928 2626 0 0 1328 1512 Sphingosine 50853 67162 44188 45354 52822 56176 9590 9736 13308 12862 12120 13316 18996 18200 12090 14834 16084 17472 28686 25246 16520 16034 15920 14290 Stearic acid 1456 1326 1280 1360 1380 1470 1504 1302 1644 1526 1828 1572 1234 1364 1191 1218 1338 1350 1168 1278 1294 1208 1652 1302 Succinylacetone 14740 16462 16036 16296 13464 12982 14004 11994 9648 10612 9836 11674 12098 12404 9494 8568 13676 13086 19920 21232 5414 5346 8710 8776 Threonic acid 104704 115452 86514 101954 129786 128600 66274 63360 55702 66328 68042 62200 59421 58920 75420 73620 82686 74626 31656 32646 44605 47878 46980 45710 Trimethylamine 1804 2322 2110 2100 2108 2316 1668 1094 1668 2240 1624 1248 1230 1402 2244 1540 2162 2794 778 742 1040 1318 576 812 Trimethylamine N-oxide 0 234 920 752 0 446 1416 1698 1732 2710 2990 2258 256 0 0 0 406 0 0 0 0 0 0 0 Xanthine 25844 27688 16660 18172 26262 27480 22892 22058 21118 24990 20650 20286 32492 35530 33302 34480 27104 31780 10782 13086 16722 15496 12892 13984 Xanthosine 21120 23778 23226 23430 27010 27828 16180 15468 15722 17626 14888 19256 21804 20606 21512 20038 20664 19192 6912 7920 8824 9460 8560 8766 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT [min] m/z meas. 17-Hydroxyprogesterone 0.3 331.22456 2'-Deoxyguanosine 5'-monophosphate 6.2 348.07068 2-Ethyl-2-hydroxybutyric acid 5.86 133.0859 2-Hydroxyadenine 1.8 152.05672 2-Phenylaminoadenosine 10.97 381.13107 2-Phenylbutyric acid 10 165.09108 2-Pyrrolidinone 6.86 86.06028 3,4-Dihydroxyphenylglycol 2.21 171.06288 3,5-Diiodo-L-tyrosine 17.32 433.88026 3-Nitrotyrosine 1.77 227.06649 3-Phenylbutyric acid 9.92 165.09111 3a,6b,7b-Trihydroxy-5b-cholanoic acid 7.18 409.29251 5'-Methylthioadenosine 5.83 298.09691 5-Methoxytryptophol 9.69 192.10217 7,8-Dihydrobiopterin 1.69 240.1092 Acetoin 5.86 89.05987 Acetone 10.84 59.04932 Adenine 5.83 136.06177 Adenosine 4.54 268.10407 Adenosine 3',5'-diphosphate 2.21 428.03679 Adenosine monophosphate 5.34 348.07117 Adenosine triphosphate 1.93 508.00327 ADP 5.35 428.03688 Alpha-N-phenylacetyl-L-glutamine 1.17 265.11199 Aminopterin 5.13 441.16872 Androstenedione 9.17 287.19688 Benzene 2.42 79.0542 Benzocaine 5.63 166.08499 Beta-Alanine 5.51 90.05524 Carnosine 1.71 249.0946 Chalcone 5.51 209.10163 Cytosine 4.83 112.05047 D-Glucurono-6,3-lactone 5.56 177.03289 Diacetyl 5.92 87.04423 Diethanolamine 13.41 106.08631 Ethanolamine 13.73 62.06016 Ethyl malonate 4.99 133.04954 Galactitol 1.18 183.08642 Gamma-Glutamylcysteine 6.1 251.06971 Gluconolactone 2.08 179.04857 Glutathione 2.11 308.09112 Glycerol 8.02 93.0548 Glycerophosphocholine 1.06 258.11029 Glycine 11.33 76.03946 Glycocholic acid 11.06 466.31718 Glycylproline 5 173.09228 Guanine 5.39 152.05681 Guanosine 4.72 284.09909 Guanosine 5'-diphosphate 1.86 444.03202 Hydroxyisocaproic acid 9.81 133.08596 Hyodeoxycholic acid 20.18 393.29785 Hypoxanthine 4.71 137.04573 Indole 6 118.06504 Indole-3-carbinol 9.73 148.07589 Indoleacrylic acid 6 188.07074 Inosine 4.72 269.08827 Isovalerylcarnitine 6.4 246.17021 L-Acetylcarnitine 1.88 204.12318 L-Dopa 5.29 198.07388 L-Fucose 1.19 165.07585 L-Homoserine 11.45 120.06527 L-Norleucine 9.17 132.10198 L-Phenylalanine 5.14 166.08635 L-Tryptophan 6 205.09736 Leucinic acid 6.19 133.08594 Maltitol 1.18 345.13922 Medroxyprogesterone 18.12 345.24039 Melatonin 6.56 233.1262 Methylmalonic acid 1.77 119.0352 Mevalonic acid 2.11 149.08087 N-Acetyl-L-alanine 1.29 132.06551 N-Acetylserotonin 6.08 219.11064 N-Methyl-a-aminoisobutyric acid 1.62 118.08623 N-Methyl-D-aspartic acid 1.16 148.06052 Niacinamide 3.84 123.05532 Nutriacholic acid 20.08 413.26642 Pantothenic acid 5.5 220.11836 PC(16:0/16:0) 13.04 734.56927 PC(18:1(9Z)/18:1(9Z)) 19.6 786.60054 Phenylacetaldehyde 9.65 121.06487 Phosphocreatine 12.15 212.03772 Propionic acid 7.02 75.04418 Pyridine 2.14 80.04984 Pyridoxal 4.73 168.06322 Pyridoxal 5'-phosphate 2.29 248.03221 Pyrrolidonecarboxylic acid 4.94 130.04983 Pyruvaldehyde 15.86 73.02855 Salicin 5.42 287.10708 Sebacic acid 14.6 102.06776 Spermine 0.81 203.22313 Sphinganine 12.44 302.30552 Sphingosine 12.11 282.2793 Stearic acid 0.57 285.27895 Succinylacetone 8.93 159.06539 Threonic acid 2.06 137.04531 Trimethylamine 1.87 60.08094 Trimethylamine N-oxide 9.15 76.07596 Xanthine 4.99 153.04073 Xanthosine 5 285.08311 METABOLITES_END #END