#METABOLOMICS WORKBENCH SIMR_Core_Facility_20230622_054105 DATATRACK_ID:4107 STUDY_ID:ST002755 ANALYSIS_ID:AN004472 PROJECT_ID:PR001716 VERSION 1 CREATED_ON June 27, 2023, 2:52 pm #PROJECT PR:PROJECT_TITLE Metabolomics Profiling of the Antiproliferative, Anti-migratory and PR:PROJECT_TITLE Anti-invasive Potential of Amlodipine in Lung Cancer Cells PR:PROJECT_TYPE LC-MS/MS PR:PROJECT_SUMMARY Lung cancer is still among the most leading causes of cancer-related deaths PR:PROJECT_SUMMARY across the world. Although chemotherapy is considered as a critical choice to PR:PROJECT_SUMMARY manage/limit cancer growth in lung cancer patients with early-stage and advanced PR:PROJECT_SUMMARY cancer stages, it has many limitations including, at least, the severe side PR:PROJECT_SUMMARY effects and chemoresistance. The latter is one of the considerable challenges to PR:PROJECT_SUMMARY lung cancer treatment. Therefore, identification of new alternative therapies PR:PROJECT_SUMMARY with lesser cytotoxic effects when compared to the currently used PR:PROJECT_SUMMARY chemotherapeutics is one of the current research approaches. Calcium channel PR:PROJECT_SUMMARY blockers (CCBs) are emerging as anti-cancer agents in several cancer types. Our PR:PROJECT_SUMMARY objective is to determine the cytotoxic effect of amlodipine on non-small cell PR:PROJECT_SUMMARY lung cancer (NSCLC) cells. Colorimetric MTT cell proliferation assay was used to PR:PROJECT_SUMMARY analyze cell viability following treatments with amlodipine in A549 and H1299 PR:PROJECT_SUMMARY NSCLC cell lines. ANOVA and Tukey’s multiple comparison test were used to PR:PROJECT_SUMMARY detect statistical significance. Half maximal (50%) inhibitory concentration PR:PROJECT_SUMMARY (IC50) values were obtained by applying nonlinear regression curve fit analysis. PR:PROJECT_SUMMARY To assess the effect of amlodipine on A549 and H1299 NSCLC cells migration and PR:PROJECT_SUMMARY invasion scratch wound-healing assay and cell invasion assay were used. Our PR:PROJECT_SUMMARY study revealed that amlodipine significantly reduced proliferation of cancer PR:PROJECT_SUMMARY cells in a dose-dependent fashion with half maximal (50%) inhibitory PR:PROJECT_SUMMARY concentration (IC50) values of 23 and 25.66 µM in A549 and H1299 cells, PR:PROJECT_SUMMARY respectively. Furthermore, amlodipine was able to reduce the invasiveness and PR:PROJECT_SUMMARY migration of cancer cells, both of which are hallmarks in the pathogenesis of PR:PROJECT_SUMMARY cancer, in both cell lines in a dose-dependent manner. Accordingly, our study PR:PROJECT_SUMMARY provides empirical evidence that amlodipine expresses anti-cancer effect to PR:PROJECT_SUMMARY NSCLC cells. However, additional investigations are required to further confirm PR:PROJECT_SUMMARY our results on a larger scale at the clinical level. 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 Metabolomics Profiling of the Antiproliferative, Anti-migratory and ST:STUDY_TITLE Anti-invasive Potential of Amlodipine in Lung Cancer Cells ST:STUDY_TYPE LC/MS/MS ST:STUDY_SUMMARY Lung cancer is still among the most leading causes of cancer-related deaths ST:STUDY_SUMMARY across the world. Although chemotherapy is considered as a critical choice to ST:STUDY_SUMMARY manage/limit cancer growth in lung cancer patients with early-stage and advanced ST:STUDY_SUMMARY cancer stages, it has many limitations including, at least, the severe side ST:STUDY_SUMMARY effects and chemoresistance. The latter is one of the considerable challenges to ST:STUDY_SUMMARY lung cancer treatment. Therefore, identification of new alternative therapies ST:STUDY_SUMMARY with lesser cytotoxic effects when compared to the currently used ST:STUDY_SUMMARY chemotherapeutics is one of the current research approaches. Calcium channel ST:STUDY_SUMMARY blockers (CCBs) are emerging as anti-cancer agents in several cancer types. Our ST:STUDY_SUMMARY objective is to determine the cytotoxic effect of amlodipine on non-small cell ST:STUDY_SUMMARY lung cancer (NSCLC) cells. Colorimetric MTT cell proliferation assay was used to ST:STUDY_SUMMARY analyze cell viability following treatments with amlodipine in A549 and H1299 ST:STUDY_SUMMARY NSCLC cell lines. ANOVA and Tukey’s multiple comparison test were used to ST:STUDY_SUMMARY detect statistical significance. Half maximal (50%) inhibitory concentration ST:STUDY_SUMMARY (IC50) values were obtained by applying nonlinear regression curve fit analysis. ST:STUDY_SUMMARY To assess the effect of amlodipine on A549 and H1299 NSCLC cells migration and ST:STUDY_SUMMARY invasion scratch wound-healing assay and cell invasion assay were used. Our ST:STUDY_SUMMARY study revealed that amlodipine significantly reduced proliferation of cancer ST:STUDY_SUMMARY cells in a dose-dependent fashion with half maximal (50%) inhibitory ST:STUDY_SUMMARY concentration (IC50) values of 23 and 25.66 µM in A549 and H1299 cells, ST:STUDY_SUMMARY respectively. Furthermore, amlodipine was able to reduce the invasiveness and ST:STUDY_SUMMARY migration of cancer cells, both of which are hallmarks in the pathogenesis of ST:STUDY_SUMMARY cancer, in both cell lines in a dose-dependent manner. Accordingly, our study ST:STUDY_SUMMARY provides empirical evidence that amlodipine expresses anti-cancer effect to ST:STUDY_SUMMARY NSCLC cells. However, additional investigations are required to further confirm ST:STUDY_SUMMARY our results on a larger scale at the clinical level. 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 ST:EMAIL tims-tof@sharjah.ac.ae ST:PHONE 065057656 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Amlodipine 01-01-10446 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 01-01-10446.d SUBJECT_SAMPLE_FACTORS - Amlodipine 01-02-10447 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 01-02-10447.d SUBJECT_SAMPLE_FACTORS - Amlodipine 02-01-10448 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 02-01-10448.d SUBJECT_SAMPLE_FACTORS - Amlodipine 02-02-10449 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 02-02-10449.d SUBJECT_SAMPLE_FACTORS - Amlodipine 03-01-10450 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 03-01-10450.d SUBJECT_SAMPLE_FACTORS - Amlodipine 03-02-10451 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 03-02-10451.d SUBJECT_SAMPLE_FACTORS - Amlodipine 04-01-10452 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 04-01-10452.d SUBJECT_SAMPLE_FACTORS - Amlodipine 04-02-10453 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 04-02-10453.d SUBJECT_SAMPLE_FACTORS - Amlodipine 05-01-10454 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 05-01-10454.d SUBJECT_SAMPLE_FACTORS - Amlodipine 05-02-10455 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 05-02-10455.d SUBJECT_SAMPLE_FACTORS - Amlodipine 06-01-10456 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 06-01-10456.d SUBJECT_SAMPLE_FACTORS - Amlodipine 06-02-10457 Treatment:Amlodipine RAW_FILE_NAME=Amlodipine 06-02-10457.d SUBJECT_SAMPLE_FACTORS - Control 01-01-10433 Treatment:Control RAW_FILE_NAME=Control 01-01-10433.d SUBJECT_SAMPLE_FACTORS - Control 01-02-10434 Treatment:Control RAW_FILE_NAME=Control 01-02-10434.d SUBJECT_SAMPLE_FACTORS - Control 02-01-10435 Treatment:Control RAW_FILE_NAME=Control 02-01-10435.d SUBJECT_SAMPLE_FACTORS - Control 02-02-10436 Treatment:Control RAW_FILE_NAME=Control 02-02-10436.d SUBJECT_SAMPLE_FACTORS - Control 03-01-10437 Treatment:Control RAW_FILE_NAME=Control 03-01-10437.d SUBJECT_SAMPLE_FACTORS - Control 03-02-10438 Treatment:Control RAW_FILE_NAME=Control 03-02-10438.d SUBJECT_SAMPLE_FACTORS - Control 04-01-10439 Treatment:Control RAW_FILE_NAME=Control 04-01-10439.d SUBJECT_SAMPLE_FACTORS - Control 04-02-10440 Treatment:Control RAW_FILE_NAME=Control 04-02-10440.d SUBJECT_SAMPLE_FACTORS - Control 05-01-10441 Treatment:Control RAW_FILE_NAME=Control 05-01-10441.d SUBJECT_SAMPLE_FACTORS - Control 05-02-10442 Treatment:Control RAW_FILE_NAME=Control 05-02-10442.d SUBJECT_SAMPLE_FACTORS - Control 06-01-10443 Treatment:Control RAW_FILE_NAME=Control 06-01-10443.d SUBJECT_SAMPLE_FACTORS - Control 06-02-10444 Treatment:Control RAW_FILE_NAME=Control 06-02-10444.d #COLLECTION CO:COLLECTION_SUMMARY A549 human lung adenocarcinoma cell line was obtained from American Type Culture CO:COLLECTION_SUMMARY Collection (ATCC, Manassas, VA, USA). Cells were maintained in Roswell Park CO:COLLECTION_SUMMARY Memorial Institute (RPMI-1640) media supplemented with 10% fetal bovine serum CO:COLLECTION_SUMMARY (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a humidified CO:COLLECTION_SUMMARY atmosphere with 5% CO2 at 37°C. CO:SAMPLE_TYPE Lung #TREATMENT TR:TREATMENT_SUMMARY Amlodipine was purchased from Tocris Bioscience (Bristol, UK). Amlodipine was TR:TREATMENT_SUMMARY dissolved in dimethyl sulfoxide (DMSO) to produce stock concentrations of 10, 50 TR:TREATMENT_SUMMARY and 100 mM DMSO. Of note, to minimize the cytotoxic effects of DMSO, the final TR:TREATMENT_SUMMARY concentration has never exceeded 0.1% in all treatment groups. Furthermore, the TR:TREATMENT_SUMMARY cells ere treated in a quadruplicate manner with DMSO as a positive control. #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 minutes. 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. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY amples 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 Water (0.1% Formic Acid) CH:SOLVENT_B ACN (0.1% Formic Acid) CH:FLOW_GRADIENT The gradient program was: 0–2 min, 99% A: 1% B; 2–17 min, 99–1% A: 1–99% CH:FLOW_GRADIENT B; 17–20 min, 99% B: 1% A. The flow rate was fixed at 0.25 mL/min. CH:FLOW_GRADIENT Subsequently, 20–20.1 min 99% B to 99% A; 20.1–28.5 min, 99% A: 1% B at 0.35 CH:FLOW_GRADIENT mL/min flow rate; 28.5–30 min; 99% A: 1% B at 0.25 mL/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 MS Comments the T-ReX 2D/3D workflow: MS:MS_COMMENTS For peak detection, a minimum intensity threshold of 1,000 counts is required, MS:MS_COMMENTS as well as a minimum peak duration of 7 spectra, with feature quantification MS:MS_COMMENTS determine using peak area. The file masses were recalibrated based on the MS:MS_COMMENTS external calibrant injected between 0-0.3 min. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples Amlodipine 01-01-10446 Amlodipine 01-02-10447 Amlodipine 02-01-10448 Amlodipine 02-02-10449 Amlodipine 03-01-10450 Amlodipine 03-02-10451 Amlodipine 04-01-10452 Amlodipine 04-02-10453 Amlodipine 05-01-10454 Amlodipine 05-02-10455 Amlodipine 06-01-10456 Amlodipine 06-02-10457 Control 01-01-10433 Control 01-02-10434 Control 02-01-10435 Control 02-02-10436 Control 03-01-10437 Control 03-02-10438 Control 04-01-10439 Control 04-02-10440 Control 05-01-10441 Control 05-02-10442 Control 06-01-10443 Control 06-02-10444 Factors Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Amlodipine Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control 1-Methyladenosine 1210 1106 940 1594 364 480 1152 1214 1328 884 674 532 976 966 584 704 534 844 654 1504 488 552 480 476 1-Methylguanine 1232 954 818 818 0 0 296 0 150 348 0 218 0 0 0 0 0 0 194 0 0 0 0 0 11a-Hydroxyprogesterone 15924 11976 15430 18026 18714 18114 18678 19898 20228 18504 20974 17822 12852 13154 15058 16660 17294 14378 15392 15714 20928 17648 15970 16860 2'-Deoxyguanosine 5'-monophosphate 1100 1196 814 1072 346 360 648 454 806 496 750 584 0 0 228 92 140 260 168 206 0 196 0 0 2,3-Butanediol 3120 2306 1600 2068 944 986 2210 2116 2418 2420 1934 2060 2234 2768 2680 2326 2166 2038 2398 2210 1918 1394 1608 2584 2-Ethyl-2-hydroxybutyric acid 116068 108332 79938 81946 47858 54148 85716 88632 90602 95750 79648 81744 108394 111316 104248 108628 81648 85292 89978 89394 71998 67552 81386 112130 2-Hydroxyadenine 6164 5502 4340 4624 4036 4150 6186 5330 3374 3230 2066 2610 2862 3390 3104 2620 2952 2580 3944 3880 1926 2062 2022 2832 2-Methyl-3-ketovaleric acid 9806 9528 7840 7886 12738 10110 10896 9716 8132 8484 2206 1776 10034 9698 9604 10168 8358 9288 8920 9152 8424 8112 11054 9732 2-Pyrrolidinone 750 374 262 504 522 414 446 544 144 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3-Hexenedioic acid 478 540 418 502 390 620 386 458 378 460 396 384 190 196 548 326 246 204 280 368 302 344 378 364 3-Phenylbutyric acid 32594 33796 34758 32576 34854 36914 35544 33402 32652 33236 19664 18786 32648 32028 35484 34660 33672 31996 34034 31740 35056 34340 33254 33290 3a,6b,7b-Trihydroxy-5b-cholanoic acid 338 552 406 300 508 406 544 454 320 394 0 0 378 574 370 0 422 410 486 474 310 298 446 348 4-Guanidinobutanoic acid 306 362 0 332 264 436 440 244 342 366 0 184 478 276 630 342 532 216 332 1236 286 632 520 368 5'-Methylthioadenosine 20200 19476 20326 23902 8260 9820 19728 22918 18330 17324 17958 21810 8406 8106 10844 9864 11146 12030 17932 15722 12904 13204 15408 19502 5-Aminolevulinic acid 800 402 524 600 376 194 244 320 182 282 172 420 248 206 222 316 298 288 422 338 522 290 182 0 5-Hydroxy-L-tryptophan 1020 704 670 820 364 390 1046 770 560 526 540 568 502 374 764 502 644 698 744 876 536 452 660 846 5-Methoxytryptophol 0 876 520 612 0 498 898 866 598 264 300 234 794 912 1084 920 224 542 304 444 880 0 862 1484 5-Methylcytidine 0 816 0 0 1098 1182 994 0 586 704 692 280 400 980 580 304 198 432 0 172 434 394 0 0 6-(Methylamino) purine 688 1110 1082 696 564 542 896 856 576 364 392 314 238 380 0 156 0 0 360 254 0 0 0 0 7-Methylguanine 11240 9942 7884 8026 3928 3722 8552 8668 5912 5772 2510 2766 1736 1566 1172 1382 990 756 1628 1574 1218 1146 924 1444 8,11,14-Eicosatrienoic acid 704 714 596 500 654 620 660 606 878 744 704 584 1070 800 688 738 1000 710 1110 760 642 710 800 744 Acetoin 175744 213088 110706 117438 59796 69664 139448 147592 166980 135208 120588 0 2724 148384 135768 169458 159672 102026 183184 179652 83158 116314 142752 203790 Acetone 1104 1708 1218 1476 1370 1086 1548 1418 1638 932 1424 938 1092 1488 1578 1812 1940 1074 1216 940 2172 1216 1088 1004 Adenine 5914 4988 5870 5672 3142 2850 6478 6070 5214 4002 5570 5978 2304 2948 3288 2716 2818 3546 4402 4744 3460 4370 4866 5558 Adenosine 8690 7130 8794 8080 0 0 4616 1822 4962 7516 0 5346 8626 0 9776 9440 5922 6114 5940 6252 4456 242 100 6220 Aldosterone 610 738 618 478 728 692 540 782 260 0 0 0 710 846 844 1008 266 482 538 638 334 576 614 518 Androstenedione 1222 404 670 1264 554 1336 564 1364 1170 1218 1672 1240 1268 1318 1278 576 1098 624 484 934 560 186 1188 1500 Aspartyl-lysine 688 548 540 694 502 564 1044 0 718 554 0 0 452 678 664 0 452 0 644 592 564 538 580 536 Asymmetric dimethylarginine 1472 1538 1770 1876 1162 1162 1384 1208 1278 1090 986 1342 1442 1314 1062 1116 2000 1634 1814 1504 1142 1714 1608 1826 Azelaic acid 770 712 0 604 590 546 772 690 274 372 0 0 1188 752 1174 1090 388 700 0 594 508 0 826 854 Benzoic acid 2176 2292 2344 1846 0 0 1074 0 0 1456 0 236 0 188 1574 1348 1870 1818 1926 1914 2206 216 0 2038 Citramalic acid 742 658 908 754 430 268 562 1140 534 392 484 0 0 0 0 0 0 0 0 0 0 0 0 0 Cytosine 1230 1368 0 410 454 0 382 386 402 448 0 790 450 0 446 504 282 442 546 280 252 0 0 260 D-Alpha-aminobutyric acid 1660 1996 1744 1846 1420 1636 1408 1946 1550 1614 1642 1824 1492 1598 1574 1456 1460 1688 1450 1660 1286 1332 1710 1888 D-Arginine 1142 930 1058 1210 1154 802 1050 1094 904 1032 780 1226 810 810 766 920 632 764 1200 986 880 852 1526 1246 D-Lysine 724 552 644 672 554 602 586 634 634 682 564 406 290 0 532 654 944 550 758 788 892 882 734 870 D-Serine 1100 1036 4106 920 566 522 666 752 130 240 0 158 728 738 882 506 652 624 966 832 466 510 1004 3162 Deoxyinosine 588 0 388 648 0 272 350 362 312 286 0 248 572 350 454 544 458 452 562 420 570 396 670 610 DGTP 882 794 908 0 1022 1160 1100 858 910 846 0 0 0 874 832 976 806 892 802 850 732 714 1116 944 Diacetyl 13596 13780 12214 13002 15078 13428 15314 14030 12188 14716 5770 5378 14692 13536 12436 12854 12070 12728 14380 13468 13146 12158 14382 13564 DL-2-aminooctanoic acid 762 824 776 620 658 586 590 772 566 800 552 764 318 196 270 512 346 388 630 610 474 450 664 782 DL-Homocystine 566 878 548 802 552 0 586 598 372 574 0 0 904 858 822 738 650 380 696 638 652 314 824 542 Elaidic acid 1294 1476 1190 1006 2154 1446 1548 1244 0 1238 392 254 934 996 876 808 1264 978 1062 1178 798 586 1028 996 Ethanolamine 772 552 570 246 660 572 0 582 250 364 294 0 978 862 352 0 256 522 762 320 440 356 728 650 Gamma-Caprolactone 532 1796 456 438 0 252 390 358 704 310 424 374 400 0 498 346 446 350 188 460 298 344 550 450 Gamma-Glutamylcysteine 436 370 356 420 312 254 504 330 322 398 240 134 0 242 332 296 870 0 554 602 486 418 0 446 Glucosamine 8196 7862 5604 6414 2898 3364 7422 6490 4382 3882 1926 1652 1078 1158 926 964 670 768 962 1570 692 632 858 828 Glutathione 0 0 264 0 0 0 190 194 308 226 724 0 0 0 0 0 0 0 556 622 926 794 556 412 Glycerophosphocholine 1144 1754 1386 1534 1232 1126 1156 1518 992 964 776 1044 1084 1448 1138 2118 1400 1620 1488 2314 1392 1554 2220 2410 Glycine 276 268 154 476 0 0 324 208 0 214 558 476 224 352 348 0 228 356 666 732 2066 2076 616 796 Glycylproline 656 0 562 414 158 386 452 240 420 522 484 402 634 600 472 542 758 726 826 508 816 1066 1036 778 Guanosine 52752 51954 45794 50130 12486 1820 45878 38170 37618 36854 11848 26276 26722 6338 28088 29528 29920 30580 33038 34586 27338 13812 13204 37004 Hippuric acid 412 580 462 514 366 540 0 558 0 480 452 572 318 270 180 248 326 494 336 500 624 540 526 574 Hydrocinnamic acid 428 0 286 532 488 392 620 600 572 574 0 0 400 330 302 390 436 304 376 520 228 366 308 420 Hydroxyisocaproic acid 1056 936 484 952 946 0 978 948 936 1300 1180 1076 1032 950 1026 0 808 1158 1130 908 0 0 1234 1022 Hydroxyoctanoic acid 1680 0 1166 1160 902 1206 1558 1260 1354 1674 1358 1656 1378 1936 1540 1612 1352 1508 1698 1466 1094 1366 1264 1684 Hypoxanthine 87432 86024 85272 87738 26248 1334 69940 57122 66070 68216 31962 61298 41784 20554 45824 50418 65474 64154 72344 75822 65558 37928 37674 83976 Indole 5306 4520 4948 6930 3460 3080 4988 5182 4596 3188 3024 4022 2486 2600 2196 2694 3910 3976 3576 4066 4052 3532 4070 5752 Indoleacetic acid 220 0 0 0 420 238 232 222 136 0 0 0 0 0 306 0 620 502 468 338 608 442 566 740 Indoleacrylic acid 31992 32044 37122 40556 22796 22302 40020 37040 28774 29208 28504 27218 14214 14438 18370 16580 23854 24814 28638 24444 24570 23208 23036 34352 Inosine 25220 20470 22470 27506 7598 1944 18206 16742 17270 18250 9714 16632 9436 5938 13010 13050 17626 17356 21820 22282 19402 10314 10462 25054 Isovalerylcarnitine 17814 16956 18378 18064 9152 9190 13084 16394 16614 15158 17566 17530 5746 5026 8240 7602 9334 8830 12904 12844 14812 17294 13022 15640 L-Acetylcarnitine 4926 4340 5188 5904 2752 3046 4930 4640 3938 4304 5132 4130 2376 2146 2554 2766 3118 3862 2954 2910 2766 2534 2970 3470 L-Alpha-aminobutyric acid 1586 1156 1088 1620 946 1008 1146 1302 0 1072 900 752 1558 1188 1426 1606 1162 1070 1098 4476 1162 1064 1300 1406 L-Aspartic acid 372 496 506 428 426 670 366 452 438 520 222 414 386 544 514 412 382 380 672 424 380 428 338 402 L-Aspartyl-L-phenylalanine 0 0 2954 928 442 412 0 3184 412 0 0 0 678 642 498 528 728 904 750 974 658 578 894 1172 L-Carnitine 2286 2484 2614 2746 1746 2170 2176 2154 2158 2046 2116 1586 1696 1188 1166 1412 1600 1628 1884 1810 1940 1764 1642 1888 L-Cystine 374 478 420 258 514 520 514 654 456 486 184 0 256 228 560 380 378 450 406 462 466 374 598 370 L-Dopa 550 426 572 532 422 220 440 540 382 464 444 456 546 562 410 518 482 614 370 390 532 528 670 454 L-Glutamic acid 1630 1488 1548 1206 686 784 1132 1366 2406 1762 1418 1786 2794 2610 2916 3946 3268 3008 2318 2176 4204 3000 1820 2926 L-Glutamine 1242 1178 1416 1328 640 830 760 1086 1368 1396 940 714 1366 1130 1344 1668 1440 1546 1322 1414 0 942 1146 1230 L-Leucine 4734 4992 4840 5624 290 0 2084 1308 2596 3120 1096 3632 1564 242 1966 2618 2762 3120 3230 2914 2600 1098 0 3286 L-Malic acid 7452 7908 5898 5902 1682 168 5614 4044 4434 4120 2028 3262 2950 1666 3174 3282 3356 3448 4588 3776 3362 1666 1690 5024 L-Methionine 1424 1450 1370 1274 812 1200 1480 1876 950 998 1842 792 514 986 690 990 886 1024 1094 1128 1188 1192 1036 1062 L-Norleucine 828 788 936 1294 246 510 722 1000 560 628 540 780 324 434 292 442 406 558 730 362 272 372 652 482 L-Phenylalanine 9346 9220 10818 9896 3640 4254 9094 8770 8136 7672 8054 9056 5968 4644 5574 6388 7916 7280 7366 7432 6574 5348 6200 9890 L-Proline 19254 18990 18642 17050 22964 22626 23464 22474 16030 17758 1752 1552 376 488 210 314 270 264 474 140 384 592 424 372 L-Tryptophan 5946 4468 5906 6230 4140 3724 7222 6006 4174 4304 5544 4610 2740 3056 2804 2968 3826 3648 4152 4434 4526 3646 3620 5906 Leucinic acid 562 542 610 512 656 698 476 968 532 528 342 750 572 690 640 686 646 590 632 474 504 506 514 458 m-Coumaric acid 3770 3534 4524 3750 0 0 1714 1302 1860 1804 0 2276 1336 0 3024 3324 3468 3852 4256 4100 3370 0 0 3458 Medroxyprogesterone 512 508 546 406 636 690 532 0 0 452 654 726 318 518 576 526 494 482 466 434 592 436 564 498 Methylmalonic acid 2446 0 3512 222 2472 3440 2072 3846 1800 0 2558 1954 0 2670 0 0 2010 1888 2578 1512 0 2130 0 0 Methylsuccinic acid 0 522 700 606 536 394 492 896 720 300 430 304 468 0 522 442 708 754 758 1158 650 622 690 708 Mevalonic acid 998 852 1004 1086 576 612 752 856 1180 898 1578 1628 774 1244 1052 1230 1276 1278 1314 1232 984 1652 1092 876 N,N-Dimethylformamide 392 520 328 334 478 944 546 0 292 208 1394 226 0 1364 516 516 514 578 506 520 448 734 194 360 N-Acetyl-D-glucosamine 748 704 766 1066 784 680 812 916 1182 800 884 684 164 0 0 0 844 872 1424 1018 1048 1014 1042 1142 N-Acetyl-L-alanine 3818 4280 3914 4102 2906 3226 3718 3698 4506 4314 2732 3264 4114 2940 3878 44552 3058 3772 3354 3750 3152 4068 3068 3352 N-Methyl-a-aminoisobutyric acid 596 434 550 810 300 272 528 522 588 528 536 468 380 346 304 334 320 372 524 644 344 468 582 482 N-Methyl-D-aspartic acid 728 598 818 690 516 752 676 800 474 622 208 386 410 428 324 506 432 536 534 588 688 560 728 934 N-Methylnicotinamide 566 640 778 720 560 1056 716 710 684 466 592 594 332 332 514 270 484 478 496 522 492 776 494 564 Niacinamide 7032 8368 6952 7282 5934 5980 5946 6718 6088 5742 7360 4704 2476 3760 2812 3734 4246 5740 4778 5284 5080 4674 3410 5138 Nutriacholic acid 13262 10372 16282 16664 11968 12146 11074 8766 8634 7918 2466 4338 26712 11936 10340 7552 9474 9058 8546 16848 14184 9386 11306 8632 o-Tyrosine 6364 6396 6762 6162 0 0 2278 1584 1400 3484 276 248 1672 0 3552 3728 5710 5390 4936 5088 3976 0 0 5940 Oleic acid 0 260 432 0 716 0 750 498 644 438 0 488 0 532 530 436 560 634 566 392 650 0 400 0 PC(16:0/16:0) 612 742 754 786 0 730 506 574 762 898 1412 0 706 568 0 670 682 668 512 742 0 0 504 586 PC(18:1(9Z)/18:1(9Z)) 24202 20620 23502 24362 16604 17198 14668 15848 19444 17514 0 64034 21914 20180 21928 21712 24466 23280 20648 23474 23214 26496 19298 21796 Perillic acid 2722 3576 2938 2680 2800 3454 2332 3278 2342 3240 1358 1446 2470 2664 3054 2488 3096 2744 5018 2848 2980 2166 3110 2786 Phenylacetaldehyde 18448 20140 20356 19824 19802 20000 17724 22234 19310 18970 4268 8992 19132 19316 19872 21288 19766 20138 19974 19636 20034 22122 19634 20998 Phenylacetic acid 1290 1368 1242 1488 0 0 998 884 1270 1294 446 1022 0 4508 694 0 704 0 1180 1074 1220 550 0 1196 Phenylpropiolic acid 1170 1234 1438 1356 0 0 784 0 0 0 0 1320 0 0 0 990 1206 1304 1512 1210 920 0 0 1392 Pregnenolone 5722 4076 3384 4090 5516 4630 5066 4838 4840 4546 2398 3040 4642 4412 5024 4320 5290 5080 5222 5396 5106 4732 5390 4370 Propionic acid 350 480 162 418 534 326 226 264 530 376 270 418 590 534 642 408 622 516 442 378 250 354 252 436 Pyridine 5850 4474 4404 5920 3248 4134 3370 4126 3854 3248 7066 3112 1314 2356 2420 2794 3152 3116 3534 3002 2804 2910 2744 2910 Pyroglutamic acid 574 1024 766 474 282 356 428 552 734 658 2444 2442 420 582 386 438 714 624 1814 1768 8070 9152 2386 3388 Pyrrolidonecarboxylic acid 760 632 504 510 204 420 374 410 668 500 344 438 656 494 650 496 890 930 1048 746 540 524 684 738 Pyruvaldehyde 2592 2756 3506 2932 2886 2724 2268 3032 2792 2904 1634 1692 2748 3406 2992 3764 3132 2732 3966 2666 2874 3526 3448 2460 Quinaldic acid 662 522 756 638 0 0 292 252 218 780 0 888 438 0 332 524 752 890 846 646 482 256 0 722 Saccharopine 1148 1338 772 770 342 564 588 792 764 512 176 732 1068 1056 1332 966 642 494 610 824 868 902 900 1176 Sebacic acid 11186 10418 6328 11534 2362 5640 8664 0 10888 11118 8582 7310 11214 10538 10904 9572 7110 7118 7798 9614 6192 5774 7088 11170 Spermine 10592 10780 12086 12218 4106 7162 12388 11378 15342 8662 8790 4622 9116 11608 8270 8958 11614 11604 12640 11030 6922 7714 17748 10048 Sphinganine 1856 2174 1728 2078 2092 1680 1560 1160 0 0 352 0 2864 2058 2112 1894 0 0 0 2228 1072 0 2442 0 Sphingosine 724 504 468 0 0 730 770 448 366 342 252 0 0 0 370 390 454 432 336 324 428 384 674 0 Stearic acid 848 712 706 776 642 956 840 1036 1050 890 1308 548 668 856 656 0 680 0 910 846 852 900 766 650 Succinylacetone 328 248 360 238 442 558 396 596 528 418 0 0 500 420 536 448 470 512 586 404 294 342 280 466 Taurodeoxycholic acid 2210 1920 1962 2012 2110 2300 2068 2186 2060 1924 0 1240 2868 2600 2310 2538 2420 2578 2362 2232 2508 0 2512 2016 Terephthalic acid 1170 962 1188 1126 1826 1826 1952 1428 576 536 0 0 1568 1044 1010 0 872 0 0 882 620 556 1188 860 Testosterone 462 310 322 466 560 346 420 510 540 364 176 0 300 410 410 382 398 480 308 248 252 382 382 540 Thymine 1438 1142 912 868 570 322 758 782 624 774 324 326 342 302 206 308 290 424 368 498 456 184 234 0 Tiglylglycine 1292 1080 1068 874 632 572 880 1056 756 730 860 878 1028 1122 1148 1408 934 730 1370 882 776 738 1098 1296 Traumatic acid 0 174 122 248 216 180 0 0 0 0 286 344 436 616 512 530 482 302 534 350 444 338 674 354 Trimethylamine 780 1024 1160 1176 1072 878 1114 1016 906 662 680 752 734 712 772 1008 704 816 742 826 738 948 820 724 Tryptophanol 664 606 1576 1566 372 322 454 398 1248 2062 1182 1174 1386 1104 910 1452 798 1054 1472 1258 1498 2182 1134 1790 Uracil 4552 5084 5430 3864 0 0 2330 602 2140 1594 0 3620 1410 0 2084 3484 2954 2488 3026 3424 2840 0 0 3670 Urea 354 366 376 552 542 318 334 402 312 130 306 322 600 478 938 468 536 700 528 538 768 546 442 304 Uridine 2352 2604 2420 2140 2410 2320 2426 2424 2046 2378 980 942 3290 3122 2756 2786 1792 2620 2238 2502 3422 3670 2756 3276 Xanthine 2250 2494 3884 3938 2714 1812 5008 4494 2194 2514 2580 2220 2128 2158 3310 2960 5226 5774 6548 6808 4600 3642 4140 5554 Xanthosine 1264 1166 2066 2132 1486 1140 2368 2304 1392 1326 980 1046 1340 974 1714 1328 2738 2694 3508 3298 2028 1986 2246 2944 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT [min] m/z meas. M meas. 1-Methyladenosine 5.15 282.1197 281.11243 1-Methylguanine 3.41 166.07243 165.06516 11a-Hydroxyprogesterone 0.31 331.22496 330.21768 2'-Deoxyguanosine 5'-monophosphate 5.02 348.0705 347.06322 2,3-Butanediol 8.06 91.07548 90.06821 2-Ethyl-2-hydroxybutyric acid 8.01 133.08602 132.07875 2-Hydroxyadenine 1.84 152.05674 151.04946 2-Methyl-3-ketovaleric acid 14.39 131.07043 130.06316 2-Pyrrolidinone 12.29 86.06075 85.05348 3-Hexenedioic acid 1.79 145.04956 144.04228 3-Phenylbutyric acid 15.44 165.091 164.08373 3a,6b,7b-Trihydroxy-5b-cholanoic acid 14.93 409.29527 408.28799 4-Guanidinobutanoic acid 13.3 146.09324 145.08596 5'-Methylthioadenosine 5.81 298.09701 297.08974 5-Aminolevulinic acid 4.36 132.06586 131.05858 5-Hydroxy-L-tryptophan 5.33 221.09235 220.08508 5-Methoxytryptophol 9.56 192.10259 191.09531 5-Methylcytidine 2.07 258.10914 257.10186 6-(Methylamino) purine 1.59 150.07757 149.07029 7-Methylguanine 5.12 166.07244 165.06517 8,11,14-Eicosatrienoic acid 0.87 307.26084 306.25356 Acetoin 8.09 89.05985 88.05258 Acetone 0.31 59.04924 58.04196 Adenine 5.81 136.06178 135.05451 Adenosine 4.56 268.10419 267.09691 Aldosterone 12.28 361.19886 360.19159 Androstenedione 0.31 287.19795 286.19067 Aspartyl-lysine 12.35 262.13766 261.13038 Asymmetric dimethylarginine 1.31 203.15038 202.1431 Azelaic acid 12.75 189.11239 188.10511 Benzoic acid 3 123.04404 122.03676 Citramalic acid 5.15 149.04561 148.03834 Cytosine 4.53 112.05063 111.04335 D-Alpha-aminobutyric acid 1.07 104.0705 103.06322 D-Arginine 1.16 175.11905 174.11177 D-Lysine 1.95 147.11292 146.10564 D-Serine 6.14 106.04983 105.04255 Deoxyinosine 4.9 253.09321 252.08593 DGTP 14.31 508.0064 506.99912 Diacetyl 15.4 87.04429 86.03702 DL-2-aminooctanoic acid 1.6 160.13338 159.12611 DL-Homocystine 13.52 269.06121 268.05393 Elaidic acid 17.72 283.26388 282.25661 Ethanolamine 10.72 62.06032 61.05304 Gamma-Caprolactone 8.47 115.07554 114.06826 Gamma-Glutamylcysteine 2.26 251.06953 250.06225 Glucosamine 5.49 180.08821 179.08093 Glutathione 4.97 308.09085 307.08357 Glycerophosphocholine 1.26 258.11013 257.10285 Glycine 4.94 76.03941 75.03213 Glycylproline 5.22 173.09227 172.085 Guanosine 4.74 284.09908 283.0918 Hippuric acid 6.98 180.06577 179.0585 Hydrocinnamic acid 10.7 151.0756 150.06832 Hydroxyisocaproic acid 5.4 133.0857 132.07843 Hydroxyoctanoic acid 8.23 161.11744 160.11016 Hypoxanthine 4.73 137.04581 136.0385 Indole 5.96 118.06502 117.05774 Indoleacetic acid 10.13 176.07076 175.06348 Indoleacrylic acid 5.97 188.07076 187.06348 Inosine 4.73 269.08819 268.08087 Isovalerylcarnitine 6.35 246.17032 245.16304 L-Acetylcarnitine 1.81 204.12313 203.11585 L-Alpha-aminobutyric acid 14.24 104.07073 103.06346 L-Aspartic acid 1.36 134.04479 133.03752 L-Aspartyl-L-phenylalanine 5.95 281.11279 280.10552 L-Carnitine 1.25 162.11258 161.10531 L-Cystine 1.28 241.03114 240.02386 L-Dopa 5.26 198.07491 197.06763 L-Glutamic acid 4.95 148.06049 147.05321 L-Glutamine 6.3 147.07673 146.06945 L-Leucine 3.03 132.10195 131.09467 L-Malic acid 4.72 135.0298 134.02252 L-Methionine 2.07 150.05778 149.0505 L-Norleucine 6.06 132.10198 131.09471 L-Phenylalanine 5.07 166.0864 165.07912 L-Proline 14.14 116.07089 115.06361 L-Tryptophan 5.96 205.09734 204.09006 Leucinic acid 0.49 133.08591 132.07863 m-Coumaric acid 3 165.05475 164.04748 Medroxyprogesterone 5.67 345.24019 344.23291 Methylmalonic acid 2.06 119.03504 118.02777 Methylsuccinic acid 5 133.04954 132.04227 Mevalonic acid 2.12 149.08092 148.07365 N,N-Dimethylformamide 2.13 74.06016 73.05289 N-Acetyl-D-glucosamine 1.33 222.09737 221.09009 N-Acetyl-L-alanine 1.37 132.06546 131.05819 N-Methyl-a-aminoisobutyric acid 1.6 118.0862 117.07893 N-Methyl-D-aspartic acid 1.66 148.06044 147.05316 N-Methylnicotinamide 1.24 137.07092 136.06364 Niacinamide 2.1 123.05523 122.04795 Nutriacholic acid 19.71 413.26653 390.27735 o-Tyrosine 3.01 182.0813 181.07403 Oleic acid 0.45 283.2634 282.25613 PC(16:0/16:0) 20.3 734.56959 733.56231 PC(18:1(9Z)/18:1(9Z)) 17.6 786.60186 785.59458 Perillic acid 15.37 167.10701 166.09974 Phenylacetaldehyde 15.44 121.06492 120.05764 Phenylacetic acid 4.74 137.06026 136.05298 Phenylpropiolic acid 3.03 147.04409 146.03682 Pregnenolone 15.4 317.24759 316.24031 Propionic acid 1.32 75.04438 74.0371 Pyridine 2.09 80.04965 79.04237 Pyroglutamic acid 5.03 130.04992 129.04265 Pyrrolidonecarboxylic acid 6.31 130.04996 129.04268 Pyruvaldehyde 15.49 73.02878 72.0215 Quinaldic acid 3.93 174.05498 173.0477 Saccharopine 1.5 277.13937 276.13209 Sebacic acid 7.94 102.06764 202.12073 Spermine 0.82 203.22315 202.21588 Sphinganine 12.33 302.30641 301.29913 Sphingosine 17.57 300.2903 299.28302 Stearic acid 14.96 285.27916 284.27188 Succinylacetone 17.9 159.06518 158.0579 Taurodeoxycholic acid 13.75 500.29936 499.29209 Terephthalic acid 19.72 149.02342 166.02675 Testosterone 12.29 289.21628 288.209 Thymine 4.94 127.05038 126.04311 Tiglylglycine 1.25 158.08125 157.07398 Traumatic acid 9.4 229.14368 228.13641 Trimethylamine 1.18 60.0809 59.07362 Tryptophanol 5.69 162.09145 161.08417 Uracil 3.05 113.03448 112.0272 Urea 15.56 61.03963 60.03235 Uridine 12.51 245.07873 244.07145 Xanthine 4.99 153.04077 152.03349 Xanthosine 5.01 285.08325 284.07597 METABOLITES_END #END