#METABOLOMICS WORKBENCH SIMR_Core_Facility_20220831_002939 DATATRACK_ID:3440 STUDY_ID:ST002273 ANALYSIS_ID:AN003715 PROJECT_ID:PR001454 VERSION 1 CREATED_ON September 1, 2022, 12:04 am #PROJECT PR:PROJECT_TITLE Multi-Omics analysis revealed a significant alteration of critical metabolic PR:PROJECT_TITLE pathways due to sorafenib-resistance in Hep3B cell lines PR:PROJECT_TYPE MS-comparative metabolomic analysis PR:PROJECT_SUMMARY Hepatocellular carcinoma (HCC) is the second prominent cause of PR:PROJECT_SUMMARY cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, PR:PROJECT_SUMMARY where sorafenib, a multiple target ty-rosine kinase inhibitor, is used as the PR:PROJECT_SUMMARY first line of treatment. Unfortunately, resistance to sorafenib is usually PR:PROJECT_SUMMARY encountered within six months of the treatment. Therefore, there is a critical PR:PROJECT_SUMMARY need to identify the underlying reasons for drug resistance. In the present PR:PROJECT_SUMMARY study, we investigated the proteomic and metabolomics alterations accompanying PR:PROJECT_SUMMARY to sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing PR:PROJECT_SUMMARY ultra-high-performance liquid chromatography quadrupole time of flight mass PR:PROJECT_SUMMARY spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) PR:PROJECT_SUMMARY library was used to identify the differentially abundant metabolites through PR:PROJECT_SUMMARY MetaboScape 4.0 software (Bruker). For protein annotation and identification, PR:PROJECT_SUMMARY the Uniprot proteome for Homo sapiens (Human) da-tabase was utilised through PR:PROJECT_SUMMARY MaxQuant. The results revealed that 27 metabolites and 18 proteins were PR:PROJECT_SUMMARY significantly dysregulated due to sorafenib resistance in Hep3B cells compared PR:PROJECT_SUMMARY to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and PR:PROJECT_SUMMARY phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered PR:PROJECT_SUMMARY metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial PR:PROJECT_SUMMARY superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and PR:PROJECT_SUMMARY calpain small subunit 1 were among the significantly altered proteins. The PR:PROJECT_SUMMARY findings revealed that resistant Hep3B cells demonstrated significant PR:PROJECT_SUMMARY alterations in amino acid and nucleotide met-abolic pathways, energy production PR:PROJECT_SUMMARY pathways and other pathways related to cancer aggressive-ness, migration, PR:PROJECT_SUMMARY proliferation, and drug-resistance. Joint pathway enrichment analysis unveiled PR:PROJECT_SUMMARY unique pathways, including the antifolate resistance pathway and other important PR:PROJECT_SUMMARY pathways that maintain cancer cells' survival, growth, and proliferation. PR:PROJECT_SUMMARY Collectively, the results identified po-tential biomarkers for PR:PROJECT_SUMMARY sorafenib-resistant HCC and gave insights into their role in chemotherapeutic PR:PROJECT_SUMMARY drug resistance, cancer initiation, progression, and aggressiveness, which may PR:PROJECT_SUMMARY contribute to better prognosis and chemotherapeutic outcomes. PR:INSTITUTE Sharjah Institute for Medical Research PR:DEPARTMENT Medicinal Chemistry PR:LAST_NAME Soares PR:FIRST_NAME Nelson PR:ADDRESS University of Sharjah, Sharjah, UAE PR:EMAIL nsoares@sharjah.ac.ae PR:PHONE +971 65 05 7763 #STUDY ST:STUDY_TITLE Multi-Omics analysis revealed a significant alteration of critical metabolic ST:STUDY_TITLE pathways due to sorafenib-resistance in Hep3B cell lines ST:STUDY_TYPE MS- comparative metabolomic analysis ST:STUDY_SUMMARY Hepatocellular carcinoma (HCC) is the second prominent cause of ST:STUDY_SUMMARY cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, ST:STUDY_SUMMARY where sorafenib, a multiple target ty-rosine kinase inhibitor, is used as the ST:STUDY_SUMMARY first line of treatment. Unfortunately, resistance to sorafenib is usually ST:STUDY_SUMMARY encountered within six months of the treatment. Therefore, there is a critical ST:STUDY_SUMMARY need to identify the underlying reasons for drug resistance. In the present ST:STUDY_SUMMARY study, we investigated the proteomic and metabolomics alterations accompanying ST:STUDY_SUMMARY to sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ST:STUDY_SUMMARY ultra-high-performance liquid chromatography quadrupole time of flight mass ST:STUDY_SUMMARY spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) ST:STUDY_SUMMARY library was used to identify the differentially abundant metabolites through ST:STUDY_SUMMARY MetaboScape 4.0 software (Bruker). For protein annotation and identification, ST:STUDY_SUMMARY the Uniprot proteome for Homo sapiens (Human) da-tabase was utilised through ST:STUDY_SUMMARY MaxQuant. The results revealed that 27 metabolites and 18 proteins were ST:STUDY_SUMMARY significantly dysregulated due to sorafenib resistance in Hep3B cells compared ST:STUDY_SUMMARY to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and ST:STUDY_SUMMARY phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered ST:STUDY_SUMMARY metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial ST:STUDY_SUMMARY superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and ST:STUDY_SUMMARY calpain small subunit 1 were among the significantly altered proteins. The ST:STUDY_SUMMARY findings revealed that resistant Hep3B cells demonstrated significant ST:STUDY_SUMMARY alterations in amino acid and nucleotide met-abolic pathways, energy production ST:STUDY_SUMMARY pathways and other pathways related to cancer aggressive-ness, migration, ST:STUDY_SUMMARY proliferation, and drug-resistance. Joint pathway enrichment analysis unveiled ST:STUDY_SUMMARY unique pathways, including the antifolate resistance pathway and other important ST:STUDY_SUMMARY pathways that maintain cancer cells' survival, growth, and proliferation. ST:STUDY_SUMMARY Collectively, the results identified po-tential biomarkers for ST:STUDY_SUMMARY sorafenib-resistant HCC and gave insights into their role in chemotherapeutic ST:STUDY_SUMMARY drug resistance, cancer initiation, progression, and aggressiveness, which may ST:STUDY_SUMMARY contribute to better prognosis and chemotherapeutic outcomes. ST:INSTITUTE Sharjah Institute for Medical Research ST:LAST_NAME Soares ST:FIRST_NAME Nelson ST:ADDRESS University of Sharjah, Sharjah, UAE ST:EMAIL nsoares@sharjah.ac.ae ST:PHONE +971 65 05 7763 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Not applicable SU:CELL_BIOSOURCE_OR_SUPPLIER Sigma-Aldrich (St. Louis, MO) SU:CELL_STRAIN_DETAILS Hep3B #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 - A01-01_57_1_1697 Phenotype:Parental RAW_FILE_NAME=A01-01_57_1_1697.d SUBJECT_SAMPLE_FACTORS - A01-02_57_1_1698 Phenotype:Parental RAW_FILE_NAME=A01-02_57_1_1698.d SUBJECT_SAMPLE_FACTORS - A02-01_58_1_1699 Phenotype:Parental RAW_FILE_NAME=A02-01_58_1_1699.d SUBJECT_SAMPLE_FACTORS - A02-02_58_1_1700 Phenotype:Parental RAW_FILE_NAME=A02-02_58_1_1700.d SUBJECT_SAMPLE_FACTORS - A03-01_59_1_1701 Phenotype:Parental RAW_FILE_NAME=A03-01_59_1_1701.d SUBJECT_SAMPLE_FACTORS - A03-02_59_1_1702 Phenotype:Parental RAW_FILE_NAME=A03-02_59_1_1702.d SUBJECT_SAMPLE_FACTORS - B01-01_60_1_1703 Phenotype:Resistant RAW_FILE_NAME=B01-01_60_1_1703.d SUBJECT_SAMPLE_FACTORS - B01-02_60_1_1704 Phenotype:Resistant RAW_FILE_NAME=B01-02_60_1_1704.d SUBJECT_SAMPLE_FACTORS - B02-01_61_1_1705 Phenotype:Resistant RAW_FILE_NAME=B02-01_61_1_1705.d SUBJECT_SAMPLE_FACTORS - B02-02_61_1_1706 Phenotype:Resistant RAW_FILE_NAME=B02-02_61_1_1706.d SUBJECT_SAMPLE_FACTORS - B03-01_62_1_1707 Phenotype:Resistant RAW_FILE_NAME=B03-01_62_1_1707.d SUBJECT_SAMPLE_FACTORS - B03-02_62_1_1708 Phenotype:Resistant RAW_FILE_NAME=B03-02_62_1_1708.d #COLLECTION CO:COLLECTION_SUMMARY Cell lines and culture conditions: The Hep3B cell line used in this study was CO:COLLECTION_SUMMARY purchased from Sigma-Aldrich (St. Louis, MO). Sorafenib-resistant Hep3B cells CO:COLLECTION_SUMMARY were developed from Hep3B cell lines One million Hep3B cells were seeded in T75 CO:COLLECTION_SUMMARY flasks and cultured in Dulbecco's Modified Eagle Medium (DMEM) as monolayers. CO:COLLECTION_SUMMARY The medium was sup-plemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, CO:COLLECTION_SUMMARY St. Louis, #F2442-500ML) and 1% penicillin/streptomycin (Sigma Aldrich, St. CO:COLLECTION_SUMMARY Louis, #P4333). Cultures were then incubated in 5% CO2 humidified atmosphere at CO:COLLECTION_SUMMARY 37°C for 3-4 days until 70-80% confluency was reached. Aseptic techniques were CO:COLLECTION_SUMMARY applied to avoid poten-tial contamination, and the confluency and contamination CO:COLLECTION_SUMMARY were checked routinely. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Generation of resistant Hep3B cell line: Resistant Hep3B cells were developed in TR:TREATMENT_SUMMARY clinically relevant models. In T-75 flasks, Hep3B cells were seeded overnight TR:TREATMENT_SUMMARY and incubated at 37°C. The cells were then treated with sorafenib at the 10 % TR:TREATMENT_SUMMARY inhibitory concentration (IC10) (0.4µM) of sorafenib (Biovision, Milpitas, CA, TR:TREATMENT_SUMMARY USA #BAY 43-9006) to mimic the clinically consumed low dose of chemotherapeutic TR:TREATMENT_SUMMARY drugs by a cancer patient who is then exposed to escalating doses over time. To TR:TREATMENT_SUMMARY develop resistance, the survived cells were transplanted to a new flask and then TR:TREATMENT_SUMMARY treated with sorafenib at escalating concentrations over six months. Then, TR:TREATMENT_SUMMARY sorafenib at concentration IC10 (0.4µM) was persistently retained in the TR:TREATMENT_SUMMARY culture media to ensure and maintain resistance. MTT cell viability assay was TR:TREATMENT_SUMMARY performed each month to validate the resistance behavior of the cells. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction: For metabolomics, three biological replicates were used. SP:SAMPLEPREP_SUMMARY An equal number of cells were utilized for each sample. The cells were SP:SAMPLEPREP_SUMMARY centrifuged separately at 15000 rpm for 10 min to separate the cells and SP:SAMPLEPREP_SUMMARY cell-free supernatants. The supernatants were dis-carded, and to each cell SP:SAMPLEPREP_SUMMARY pellet (~3x 106), 1 mL of 0.1% formic acid in methanol was added, followed by SP:SAMPLEPREP_SUMMARY vertexing for 2 min x 4, interrupted by 15 min incubation on ice. This is SP:SAMPLEPREP_SUMMARY followed by sonication using a COPLEY probe-sonicator (QSONICA SONI-CATOR, USA) SP:SAMPLEPREP_SUMMARY for 30 seconds with 30% amplifier in an ice bath. Subsequently, the cel-lular SP:SAMPLEPREP_SUMMARY debris of each sample was collected by centrifugation at 15000 rpm for 10 min at SP:SAMPLEPREP_SUMMARY 4°C. The supernatants were separated and dried at 37 ± 1 °C using EZ-2 Plus SP:SAMPLEPREP_SUMMARY (Gene-Vac-Ipswich, UK). Finally, 200 µL of 0.1% formic acid in water was used SP:SAMPLEPREP_SUMMARY to resuspend the dried samples, which were then vortexed for 2 min, filtered by SP:SAMPLEPREP_SUMMARY a 0.45µm hydro-philic Nylon Syringe Filter and transferred to 200 μL SP:SAMPLEPREP_SUMMARY (micro-inserts) in LC vials. The quality control (QC) sample was prepared by SP:SAMPLEPREP_SUMMARY pooling the same volume (10 μL) of each sample, and all samples were placed in SP:SAMPLEPREP_SUMMARY the autosampler at a temperature set at 4℃ and analyzed with UHPLC-QTOF-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The Elute UHPLC and Q-TOF Mass Spectrometer (Bruker, Bremen, Germany) were CH:CHROMATOGRAPHY_SUMMARY utilized for metabolite and peptide detection. The Elute HPG 1300 pumps, Elute CH:CHROMATOGRAPHY_SUMMARY Au-tosampler (Bruker, Bremen, Germany), and Hamilton® Intensity Solo 2 C18 CH:CHROMATOGRAPHY_SUMMARY column (100 mm x 2.1 mm, 1.8 m beads) were employed using reversed-phase CH:CHROMATOGRAPHY_SUMMARY chromatog-raphy. Solvents used for separation were 0.1 % FA in LC grade water CH:CHROMATOGRAPHY_SUMMARY (solvent A) and 0.1 % FA in ACN (solvent B). Each metabolite and protein extract CH:CHROMATOGRAPHY_SUMMARY were analyzed in duplicate. For metabolomics analysis, the column was kept at CH:CHROMATOGRAPHY_SUMMARY 35°C, and each sample was injected twice with an in-jection volume of 10 µL. CH:CHROMATOGRAPHY_SUMMARY Sample elution was performed in 30 min gradient starting with 1% ACN for 2 min CH:CHROMATOGRAPHY_SUMMARY and then ramped to 99% ACN within 15 min. After that, 99% ACN was kept for 3 CH:CHROMATOGRAPHY_SUMMARY min, and then the re-equilibration to 1% ACN was done for 10 min. The flow rate CH:CHROMATOGRAPHY_SUMMARY was 0.25 mL/min for 20 min and then 0.35 mL/min for 8.3 min and then the flow CH:CHROMATOGRAPHY_SUMMARY rate set at 0.25 mL/min for 1.7 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME UHPLC-QTOF-MS CH:COLUMN_NAME Hamilton® Intensity Solo 2 C18 column (100 mm x 2.1 mm, 1.8 m beads) CH:FLOW_GRADIENT Sample elution was performed in 30 min gradient starting with 1% ACN for 2 min CH:FLOW_GRADIENT and then ramped to 99% ACN within 15 min. After that, 99% ACN was kept for 3 CH:FLOW_GRADIENT min, and then the re-equilibration to 1% ACN was done for 10 min CH:FLOW_RATE was 0.25 mL/min for 20 min and then 0.35 mL/min for 8.3 min and then the flow CH:FLOW_RATE rate set at 0.25 mL/min for 1.7 min. CH:COLUMN_TEMPERATURE 35°C CH:SOLVENT_A 0.1 % FA in LC grade water CH:SOLVENT_B 0.1 % FA in ACN #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker timsTOF MS:INSTRUMENT_TYPE Trapped Ion Mobility Q-TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS For MS2 acquisition in metabolomics analysis, the collision energy was MS:MS_COMMENTS fluctuated between 100-250% of 20 eV and end plate offset of 500V. The MS:MS_COMMENTS acquisition was in two sections: auto MS scan for the calibrant sodium formate MS:MS_COMMENTS in 0-0.3 min, and auto MS/MS for fragmentation, in 0.3 to 30 min. Positive mode MS:MS_COMMENTS at 12 Hz was performed in both acquisition sections . The scan range was 20 to MS:MS_COMMENTS 1300 m/z, the precursor ion’s width of ±0.5, the precursors number of 3 , the MS:MS_COMMENTS cycle time of 0.5 s econds, and the threshold of 400 counts. After three MS:MS_COMMENTS spectra, active exclusion was performed and released after 0.2 min. A timsTOF MS:MS_COMMENTS (Bruker, Bremen, Germany) with an Apollo II electrospray ionization (ESI) source MS:MS_COMMENTS was utilized for the MS analysis with the following parameters: the nebulizer MS:MS_COMMENTS pressure was 2.2 bar, the drying gas flow rate was 10 L/min, the drying MS:MS_COMMENTS tempera-ture was 220°C, and the capillary voltage was 4500 V. In the first 0.3 MS:MS_COMMENTS min of each LC-MS/MS run, the external cali-brant, sodium formate, was injected. MS:MS_COMMENTS Mass calibration was done prior to analysis ac-cording to the manufacturer’s MS:MS_COMMENTS recommendations using external mass calibration (10 mM sodium formate calibrant MS:MS_COMMENTS solution). The performance of the column and the mass spectrometer was tested MS:MS_COMMENTS using a test mixture of (TRX-2101/RT-28-calibrants for Bruker T-ReX LC-QTOF MS:MS_COMMENTS solution from Nova Medical Testing Inc.) to check the performance of MS:MS_COMMENTS reversed-phase liquid chromatography (RPLC) separation and perform multipoint MS:MS_COMMENTS re-tention time calibration, and (TRX-3112-R/MS Certified Human serum for Bruker MS:MS_COMMENTS T-ReX LC-QTOF solution from Nova Medical Testing Inc.) to check the performance MS:MS_COMMENTS of sample preparation protocols as well as LC-MS instruments. This product is MS:MS_COMMENTS prepared from pooled human blood. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples A01-01_57_1_1697 A01-02_57_1_1698 A02-01_58_1_1699 A02-02_58_1_1700 A03-01_59_1_1701 A03-02_59_1_1702 B01-01_60_1_1703 B01-02_60_1_1704 B02-01_61_1_1705 B02-02_61_1_1706 B03-01_62_1_1707 B03-02_62_1_1708 Factors Phenotype:Parental Phenotype:Parental Phenotype:Parental Phenotype:Parental Phenotype:Parental Phenotype:Parental Phenotype:Resistant Phenotype:Resistant Phenotype:Resistant Phenotype:Resistant Phenotype:Resistant Phenotype:Resistant 1,3-Dimethyluric acid 0 0 0 0 0 0 0 0 0 0 0 2608 16-Dehydroprogesterone 0 0 0 0 0 0 0 0 0 0 0 2206 1-Butanol 2894 2446 2866 2940 2652 3048 2590 2702 3106 2736 3464 2814 2,5-Furandicarboxylic acid 10834 10248 12926 12726 2498 6584 11124 14434 5848 6772 7344 7540 2-Hydroxycaproic acid 0 0 0 0 252 0 10738 416 332 410 232 340 2-Hydroxyphenethylamine 12078 11312 1198 1138 16428 16664 12970 14282 14300 14390 12840 11826 2-Phenylaminoadenosine 2038 1436 214 116 1768 0 1232 0 2360 1922 2150 0 2-Pyrrolidinone 3270 3174 588 548 6868 5450 6020 6356 6974 6634 4294 4506 3,5-Dimethoxyphenol 2602 2304 3576 2638 1708 1766 4536 4320 7976 9002 2662 2518 3-Hexenedioic acid 898 1328 196 0 0 2058 1216 1158 1640 14466 1412 1440 3-Hydroxymethylglutaric acid 824 650 84 0 2170 2588 1840 1746 2048 1942 1930 2186 3-Methoxyphenylacetic acid 2436 2394 330 0 2926 2940 2696 2742 2796 2540 1418 1650 3-Methylindole 6474 6028 6970 6950 5102 0 11640 9912 7402 7640 10008 10230 5-Dodecenoic acid 2174 1472 614 542 716 0 884 1864 1558 1548 2024 2530 5-Hydroxytryptophol 0 0 0 0 0 0 0 0 0 0 0 21628 5-Methoxytryptophol 0 2472 0 0 0 1760 0 0 2102 0 2308 0 5'-Methylthioadenosine 10256 11000 1440 1356 46478 54226 46052 45034 38536 35716 37662 36296 5-Phenylvaleric acid 494 654 304 178 1294 1198 594 686 1896 2338 1420 1218 6-Dimethylaminopurine 3526 3696 3358 2776 2382 2066 1710 1820 1852 1628 2302 1796 6-Methyladenine 704 694 118 168 3694 3574 348 646 532 458 444 460 7-Methylguanine 1112 952 180 0 4286 3930 540 662 606 614 350 514 9-Methyluric acid 606 0 0 0 812 602 278 518 0 168 1584 2220 Acetic acid 93020 95650 107848 102994 92602 93762 98312 89524 95250 97688 104804 103558 Acetone 28272 28630 27832 27812 24890 24542 25170 26132 24562 21410 23974 21684 Adenine 544 434 248 0 1830 1488 10902 11786 10134 10236 7464 7674 Adenosine monophosphate 3460 4054 1562 1372 24660 23040 153564 164148 0 116644 91734 98698 ADP 1798 1330 246 470 6930 4790 36516 33202 35536 33870 32626 33480 All-trans-retinoic acid 0 0 0 0 0 0 0 2140 0 0 0 0 Alpha-Aspartyl-lysine 3500 2198 1194 1466 3056 0 4818 3766 3890 3284 3810 0 Aniline 4242 4426 4930 3340 2218 1926 5762 5916 3434 3396 3650 3374 Asymmetric dimethylarginine 1136 1110 288 270 3972 4168 2188 2730 2652 2432 2604 2770 Atenolol 1928 0 0 436 5168 4570 3608 3338 4446 3860 5544 4840 Benzenebutanoic acid 1088 962 484 0 2204 1308 1782 1454 1696 1640 1738 2316 Benzoic acid 2192 1548 1164 1028 2016 1836 2506 1762 1812 2386 1860 2086 Butanone 11612 12528 10174 11230 9672 8796 10150 12684 9460 10190 9298 8612 Cinnamic acid 2218 2188 464 426 7940 8758 6358 8346 8904 8896 5836 9136 Cis-Aconitic acid 1588 1652 2030 2170 1266 1130 1470 1068 1164 0 1602 1074 Citicoline 7410 7320 992 1040 31716 33280 17092 20238 16710 14622 17966 19940 Creatine 6460 6390 2162 1276 21484 23242 42680 44476 39526 29656 34572 40990 Cyclic AMP 0 150 0 0 190 350 2096 1562 1870 2064 0 1958 Cytidine 7310 6296 0 932 27784 27976 5878 6168 7432 7734 5542 5314 Cytidine monophosphate 2358 1672 554 598 4942 4874 3614 2698 2024 2538 2912 2950 Cytosine 0 772 166 270 0 1556 4712 4768 4936 4358 3864 3334 D-Alanine 1080 1652 0 0 5970 7554 11728 12286 10612 9542 10372 11002 Dehydroascorbic acid 2166 2428 2310 2810 898 0 2400 3906 1062 1410 1118 1734 Deoxyguanosine 57724 46608 8700 8450 165596 186896 280516 248996 405000 391064 349852 330724 Dextromethorphan 11302 11688 11180 10036 6222 0 29224 25440 17480 15686 16760 1008 D-Glucurono-6,3-lactone 1684 1660 1272 0 2092 1716 1412 1288 1448 0 1322 2020 Diethanolamine 2462 2098 400 452 2566 3098 2954 2094 2738 2816 2506 2296 DL-2-aminooctanoic acid 294 728 0 0 1284 2006 864 1210 1006 852 840 892 Elaidic acid 44464 9986 0 2784 3996 0 11068 21756 0 20780 20166 0 Flavin mononucleotide 0 846 108 0 2104 2230 1688 2184 2442 2672 1092 1396 Flavone 0 0 0 0 0 2242 0 218 2968 0 0 182 Glutathione 1424 1400 5520 4262 7252 7366 9642 9084 15288 15368 60522 66224 Glycerophosphocholine 4318 3274 0 0 10994 11716 39020 45970 36604 32766 33664 43258 Guaifenesin 212 310 0 288 476 608 1248 782 2020 1124 0 466 Guanidine 2374 2500 2900 2432 1924 0 1946 2032 1956 1540 1722 1846 Guanine 37894 35240 7956 7572 125002 136276 63332 69066 98044 87048 76036 70514 Guanosine 19990 18146 3034 4216 79760 83632 34422 32672 56336 49404 36864 34250 Guanosine monophosphate 280 514 524 294 2372 2174 15876 15150 9640 10458 7108 9368 Homocitrulline 0 0 0 0 0 0 0 0 0 0 2242 0 Hyodeoxycholic acid 2096 174 188 198 542 0 148 0 0 1742 1916 0 Hypoxanthine 172984 160744 34288 36068 456984 490904 239136 231364 291678 263256 231724 204240 Ibuprofen 2096 0 584 986 992 0 0 0 2020 2170 1878 0 Indole 1244 1762 1352 1554 1340 1278 2582 2878 1864 1898 2394 2616 Indoleacrylic acid 15402 18728 2620 3282 76272 77338 72812 73538 68512 54578 70842 44224 Inosine 44116 37398 9354 9874 151530 166416 57560 49442 77540 69214 49484 43472 Isobutyric acid 10566 9776 12274 10100 11862 12128 12580 15878 13458 12028 11646 12110 Isoferulic acid 2350 2058 3228 1866 1162 1308 5042 4380 8848 8158 1814 1520 Isovalerylcarnitine 0 0 0 974 0 2082 0 172 0 2912 0 0 L-Acetylcarnitine 3272 2738 468 322 21008 18178 15094 13330 13414 14296 12606 14456 L-Arginine 4516 4346 1192 1108 13446 12006 226 322 310 72 394 344 L-Aspartic acid 8068 8432 1364 1446 23510 23670 19118 18154 17232 18300 16502 15984 L-Carnitine 2882 2696 656 866 8008 8918 4990 4898 4968 4344 4446 3984 L-Dopa 1990 1876 2058 1958 1378 1470 942 1248 846 1180 1054 1030 L-Glutamic acid 3986 3572 1360 1384 7786 8424 5316 6328 5894 4676 7546 5870 Linoleic acid 2146 384 0 236 0 0 492 440 0 842 0 0 L-Malic acid 1760 1854 560 584 6304 6880 3570 3808 5474 4530 3938 3170 L-Norleucine 3992 4148 894 0 15514 16502 11386 12198 11792 11778 10556 11590 L-Phenylalanine 30690 26904 6576 5648 95314 86310 82998 96906 88588 93732 72714 80956 L-Proline 3184 3150 850 874 7798 7786 9186 9962 10224 8902 8098 9704 L-Tryptophan 4508 4180 1340 866 15472 15374 20744 19716 17730 14276 16732 10542 m-Coumaric acid 0 0 0 0 0 23086 26122 0 0 0 0 0 Medroxyprogesterone 4692 4572 376 516 7400 6400 0 0 0 4922 7262 6966 Melatonin 1610 2318 0 0 1734 2138 2380 2542 2844 2508 1702 1614 Metoprolol 11128 11586 1168 1022 14856 16584 16510 14536 19052 19746 16904 13544 N-Acetylglutamic acid 1810 2088 494 0 8414 7344 5784 5086 5182 4868 3926 4278 N-Acetyl-L-aspartic acid 12346 13564 2152 2462 33364 33722 27392 25984 22628 22282 18272 16872 N-Acetyl-L-phenylalanine 642 526 478 288 4296 4248 918 958 1810 1366 4504 4104 N-Acetylneuraminic acid 0 0 202 0 0 0 0 1978 0 0 0 3696 N-Acetylputrescine 3944 4532 1154 1030 5752 4966 5076 6594 5208 4986 3734 3560 Niacinamide 27998 23194 3284 3488 86272 93986 97482 106950 119042 96064 95524 92296 Nicotinamide ribotide 2220 2500 122 324 8538 8082 2066 1978 1794 1526 1702 1548 N-Methyl-a-aminoisobutyric acid 2284 2588 0 0 5596 6356 4352 5520 4984 5574 4388 4604 N-Methyl-D-aspartic acid 2754 2020 350 494 4968 5644 3900 3776 3654 3642 4334 4382 Norepinephrine 778 1032 0 0 3688 3898 2670 2524 3444 3076 2462 2900 Normetanephrine 1078 984 0 498 5234 6118 3916 3474 5050 3428 3416 3272 Nutriacholic acid 0 0 0 0 0 0 0 0 1984 0 3368 0 o-Tyrosine 7374 6926 1212 1096 28972 26728 26418 24780 27134 26006 21854 25488 Oxypurinol 23474 21456 3978 2968 65078 67962 27924 29814 29460 30650 25344 23168 Pantothenic acid 20010 18008 2854 3346 84970 81762 64414 66506 70090 63242 50694 42494 PC(16:0/16:0) 162 0 0 254 0 0 2238 1984 3016 2314 2898 0 PC(18:1(9Z)/18:1(9Z)) 0 0 7904 10498 13534 0 40094 159974 0 86818 0 0 Perillic acid 866 1210 810 808 990 856 1390 1432 2718 2824 1072 1356 Phenylacetaldehyde 2258 2350 2662 2790 3636 3834 3516 3644 4536 5072 3096 4086 Phenylpropanolamine 1084 1136 386 366 590 696 3184 3100 3768 3030 1622 932 Phosphoric acid 4006 3594 1854 1848 7542 7622 6454 6958 8066 6470 5758 7526 Picolinic acid 674 708 178 188 1272 1552 1920 2300 2810 2298 2174 1808 Pregnenolone 0 0 0 0 0 1258 0 0 3838 0 2718 0 Propanal 55040 55120 56612 57180 33928 36302 45354 45712 38240 38292 34020 32178 Protocatechuic acid 8116 8498 712 588 6686 7054 6532 6878 7456 6306 7430 8362 Pyridine 4472 4084 668 806 19382 20804 23514 26354 24970 24846 20584 0 Pyridoxal 5'-phosphate 1828 2046 1710 2068 1804 2164 0 0 1750 1748 1862 1662 Pyroglutamic acid 44040 49456 7674 8194 186908 182428 171826 177558 179348 195082 181230 166108 Quinaldic acid 1280 1400 254 238 6784 7546 6680 6334 7234 6536 5768 5500 Salbutamol 2850 2490 452 572 3616 3132 3162 3256 0 3802 2652 2774 Sphinganine 658 626 640 816 2988 0 434 578 448 630 456 622 Sphingosine 1208 1218 2300 2014 10452 0 1152 1322 2358 1864 1096 0 Succinic acid 304 390 0 0 980 1030 1870 2448 3546 3396 4756 4804 Tetracosanoic acid 6966 0 0 0 0 5348 0 0 19850 0 0 0 Threonic acid 0 0 0 0 45728 140548 194540 0 47662 0 19464 0 Tolnaftate 1200 1336 1386 1688 1628 1688 2712 1812 2790 2068 1936 1858 Trans-Aconitic acid 1690 1756 2132 1704 0 1920 0 1514 0 1700 1416 0 Traumatic acid 482 0 968 0 1644 0 0 1544 0 2342 1584 0 Trimethylamine 10862 12998 2454 2286 33158 30906 26642 30224 29456 26948 28504 25630 Tryptamine 0 0 0 0 0 29086 0 0 0 0 0 0 Umbelliferone 1222 2040 1292 718 1998 1770 1084 1674 1682 1748 1408 2096 Uracil 6188 6306 1004 1266 22266 22842 9752 10956 12818 11806 9738 11302 Ureidosuccinic acid 0 0 0 0 0 0 34032 0 0 0 0 10598 Uridine 7072 6022 0 0 30584 31254 10442 10454 14460 14686 10692 11986 Uridine 5'-monophosphate 528 412 154 0 2276 2158 19192 18618 16942 15510 13220 14224 Uridine diphosphate-N-acetylglucosamine 4026 3736 1224 1618 11918 11334 12768 12652 11430 10636 14114 14682 Urocanic acid 1844 3078 416 244 688 332 6368 5996 10382 9622 1046 960 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT [min] m/z meas. M meas. Ions MS/MS Molecular Formula AQ 1,3-Dimethyluric acid 11,77 197,07091 196,06364 [M+H]+ TRUE C7H8N4O3 1100 16-Dehydroprogesterone 15,31 313,21995 312,21267 [M+H]+ TRUE C21H28O2 1100 1-Butanol 22,64 75,08273 74,07545 [M+H]+ TRUE C4H10O 1120 2,5-Furandicarboxylic acid 0,84 157,01761 156,01033 [M+H]+ TRUE C6H4O5 1100 2-Hydroxycaproic acid 10,41 133,08571 132,07844 [M+H]+ TRUE C6H12O3 2100 2-Hydroxyphenethylamine 7,27 138,09106 137,08378 [M+H]+ TRUE C8H11NO 2120 2-Phenylaminoadenosine 13,71 359,14796 358,14069 [M+H]+ TRUE C16H18N6O4 1110 2-Pyrrolidinone 1,25 86,06025 85,05298 [M+H]+ TRUE C4H7NO 2220 3,5-Dimethoxyphenol 9,11 155,06978 154,0625 [M+H]+ TRUE C8H10O3 2120 3-Hexenedioic acid 7,4 145,04867 144,0414 [M+H]+ TRUE C6H8O4 1110 3-Hydroxymethylglutaric acid 1,5 163,06024 162,05296 [M+H]+ TRUE C6H10O5 2100 3-Methoxyphenylacetic acid 11,07 167,0714 166,06413 [M+H]+ TRUE C9H10O3 2110 3-Methylindole 14,57 132,08049 131,07322 [M+H]+ TRUE C9H9N 2120 5-Dodecenoic acid 17,44 199,16732 198,16004 [M+H]+ TRUE C12H22O2 2100 5-Hydroxytryptophol 10,84 178,08658 177,0793 [M+H]+ TRUE C10H11NO2 2110 5-Methoxytryptophol 10,52 192,10094 191,09366 [M+H]+ TRUE C11H13NO2 2110 5'-Methylthioadenosine 7,42 298,09683 297,08956 [M+H]+ TRUE C11H15N5O3S 2110 5-Phenylvaleric acid 11,53 179,10662 178,09934 [M+H]+ TRUE C11H14O2 2100 6-Dimethylaminopurine 1,24 164,09196 163,08469 [M+H]+ TRUE C7H9N5 2120 6-Methyladenine 4,65 150,07811 149,07083 [M+H]+ TRUE C6H7N5 2120 7-Methylguanine 4,13 166,07284 165,06557 [M+H]+ TRUE C6H7N5O 2120 9-Methyluric acid 9,31 183,05395 182,04668 [M+H]+ TRUE C6H6N4O3 1100 Acetic acid 0,34 61,02937 60,02209 [M+H]+ TRUE C2H4O2 2020 Acetone 21,46 59,04994 58,04267 [M+H]+ TRUE C3H6O 2020 Adenine 3,12 136,06046 135,05319 [M+H]+ TRUE C5H5N5 2110 Adenosine monophosphate 3,14 348,06982 347,06254 [M+H]+ TRUE C10H14N5O7P 2220 ADP 5,79 428,03576 427,02848 [M+H]+ TRUE C10H15N5O10P2 2110 All-trans-retinoic acid 14,05 301,21154 300,20427 [M+H]+ TRUE C20H28O2 1100 Alpha-Aspartyl-lysine 13,06 262,14377 261,1365 [M+H]+ TRUE C10H19N3O5 1110 Aniline 16,5 94,06526 93,05798 [M+H]+ TRUE C6H7N 2110 Asymmetric dimethylarginine 1,41 203,15009 202,14281 [M+H]+ TRUE C8H18N4O2 2210 Atenolol 8,42 267,17033 266,16305 [M+H]+ TRUE C14H22N2O3 2100 Benzenebutanoic acid 9,55 165,08657 164,07929 [M+H]+ TRUE C10H12O2 1100 Benzoic acid 13,05 123,04271 122,03543 [M+H]+ TRUE C7H6O2 2110 Butanone 0,82 73,06514 72,05786 [M+H]+ TRUE C4H8O 2220 Cinnamic acid 7,18 149,05962 148,05234 [M+H]+ TRUE C9H8O2 2120 Cis-Aconitic acid 1,95 175,01887 174,01159 [M+H]+ TRUE C6H6O6 1120 Citicoline 1,38 489,11593 488,10846 [M+H]+, [M+Na]+ TRUE C14H26N4O11P2 2120 Creatine 1,29 132,07691 131,06963 [M+H]+ TRUE C4H9N3O2 2220 Cyclic AMP 5,74 330,05896 329,05169 [M+H]+ TRUE C10H12N5O6P 2110 Cytidine 2,47 244,09275 243,08547 [M+H]+ TRUE C9H13N3O5 2110 Cytidine monophosphate 1,34 324,05978 323,05251 [M+H]+ TRUE C9H14N3O8P 2110 Cytosine 1,76 112,05049 111,04321 [M+H]+ TRUE C4H5N3O 2200 D-Alanine 1,31 90,0559 89,04862 [M+H]+ TRUE C3H7NO2 2220 Dehydroascorbic acid 0,84 175,02725 174,01997 [M+H]+ TRUE C6H6O6 1100 Deoxyguanosine 6,96 268,10347 267,09619 [M+H]+ TRUE C10H13N5O4 2120 Dextromethorphan 12,99 272,20111 271,19384 [M+H]+ TRUE C18H25NO 2120 D-Glucurono-6,3-lactone 2,14 177,03453 176,02726 [M+H]+ TRUE C6H8O6 1110 Diethanolamine 2,21 106,08631 105,07903 [M+H]+ TRUE C4H11NO2 2120 DL-2-aminooctanoic acid 1,7 160,13324 159,12597 [M+H]+ TRUE C8H17NO2 2100 Elaidic acid 18,27 283,26419 282,25716 [M+H]+, [M-H2O+H]+ TRUE C18H34O2 2210 Flavin mononucleotide 7,83 457,1139 456,10663 [M+H]+ TRUE C17H21N4O9P 1100 Flavone 16,24 223,07723 222,06995 [M+H]+ TRUE C15H10O2 2110 Glutathione 2,74 308,09093 307,08365 [M+H]+ TRUE C10H17N3O6S 2110 Glycerophosphocholine 1,23 258,11049 257,10321 [M+H]+ TRUE C8H20NO6P 2220 Guaifenesin 9,9 199,09273 198,08545 [M+H]+ TRUE C10H14O4 1100 Guanidine 0,85 60,05282 59,04554 [M+H]+ TRUE CH5N3 1100 Guanine 6,98 152,05635 151,04907 [M+H]+ TRUE C5H5N5O 2110 Guanosine 6,99 284,09873 283,09145 [M+H]+ TRUE C10H13N5O5 2120 Guanosine monophosphate 3,54 364,06485 363,05757 [M+H]+ TRUE C10H14N5O8P 2220 Homocitrulline 8,52 190,12336 189,11608 [M+H]+ TRUE C7H15N3O3 1100 Hyodeoxycholic acid 20,39 393,29499 392,28771 [M+H]+ TRUE C24H40O4 1200 Hypoxanthine 6,99 137,04539 136,03811 [M+H]+ TRUE C5H4N4O 2120 Ibuprofen 20,62 207,1353 206,12802 [M+H]+ TRUE C13H18O2 1100 Indole 14,57 118,0626 117,05533 [M+H]+ TRUE C8H7N 1120 Indoleacrylic acid 7,58 188,07067 187,06339 [M+H]+ TRUE C11H9NO2 2120 Inosine 7 269,08772 268,08045 [M+H]+ TRUE C10H12N4O5 2120 Isobutyric acid 5,77 89,05893 88,05165 [M+H]+ TRUE C4H8O2 2120 Isoferulic acid 9,11 195,06314 194,05587 [M+H]+ TRUE C10H10O4 1120 Isovalerylcarnitine 11,14 246,16664 245,15936 [M+H]+ TRUE C12H23NO4 1110 L-Acetylcarnitine 2,28 204,12307 203,1158 [M+H]+ TRUE C9H17NO4 2220 L-Arginine 1,05 175,11925 174,11197 [M+H]+ TRUE C6H14N4O2 2220 L-Aspartic acid 2,44 134,04465 133,03735 [M+H]+, [M-H2O+H]+ TRUE C4H7NO4 2220 L-Carnitine 1,2 162,1127 161,10543 [M+H]+ TRUE C7H15NO3 2220 L-Dopa 7,28 198,07574 197,06846 [M+H]+ TRUE C9H11NO4 2120 L-Glutamic acid 1,21 148,06061 147,05334 [M+H]+ TRUE C5H9NO4 2120 Linoleic acid 18,69 281,24745 280,24017 [M+H]+ TRUE C18H32O2 2200 L-Malic acid 6,99 135,02949 134,02222 [M+H]+ TRUE C4H6O5 2100 L-Norleucine 4,35 132,10137 131,09409 [M+H]+ TRUE C6H13NO2 2220 L-Phenylalanine 7,17 166,08621 165,07893 [M+H]+ TRUE C9H11NO2 2120 L-Proline 1,32 116,07063 115,06335 [M+H]+ TRUE C5H9NO2 2220 L-Tryptophan 7,58 205,09672 204,08945 [M+H]+ TRUE C11H12N2O2 2120 m-Coumaric acid 4,62 165,05234 164,04577 [M+H]+, [M-H2O+H]+, [M+H]+ TRUE C9H8O3 2220 Medroxyprogesterone 7,33 173,12319 344,23183 [M+H+H]2+ TRUE C22H32O3 2110 Melatonin 7,8 233,12852 232,12124 [M+H]+ TRUE C13H16N2O2 2110 Metoprolol 7,97 268,19051 267,18323 [M+H]+ TRUE C15H25NO3 2110 N-Acetylglutamic acid 4 212,0521 189,0629 [M+Na]+, [M+H]+, [M-H2O+H]+ TRUE C7H11NO5 2220 N-Acetyl-L-aspartic acid 2,44 198,03763 175,04815 [M+Na]+, [M+H]+ TRUE C6H9NO5 2220 N-Acetyl-L-phenylalanine 7,28 208,10031 207,09303 [M+H]+ TRUE C11H13NO3 1100 N-Acetylneuraminic acid 12,43 310,10949 309,10221 [M+H]+ TRUE C11H19NO9 1110 N-Acetylputrescine 1,26 131,11809 130,11081 [M+H]+ TRUE C6H14N2O 2220 Niacinamide 3,16 123,05473 122,04746 [M+H]+ TRUE C6H6N2O 2120 Nicotinamide ribotide 1,74 335,06432 334,05704 [M+H]+ TRUE C11H15N2O8P 2200 N-Methyl-a-aminoisobutyric acid 1,61 118,08625 117,07897 [M+H]+ TRUE C5H11NO2 2110 N-Methyl-D-aspartic acid 1,18 170,04264 147,05304 [M+Na]+, [M+K]+ TRUE C5H9NO4 2100 Norepinephrine 3,96 170,08057 169,07345 [M+H]+, [M-H2O+H]+ TRUE C8H11NO3 2220 Normetanephrine 7,3 184,09626 183,08898 [M+H]+ TRUE C9H13NO3 2110 Nutriacholic acid 16,55 391,28826 390,28099 [M+H]+ TRUE C24H38O4 1100 o-Tyrosine 4,62 182,08043 181,07315 [M+H]+ TRUE C9H11NO3 2220 Oxypurinol 4,5 153,04044 152,03316 [M+H]+ TRUE C5H4N4O2 2120 Pantothenic acid 7,3 220,1176 219,11017 [M+H]+, [M+Na]+ TRUE C9H17NO5 2120 PC(16:0/16:0) 12,99 734,57396 733,56669 [M+H]+ TRUE C40H80NO8P 1000 PC(18:1(9Z)/18:1(9Z)) 18,08 786,59801 785,59067 [M+H]+, [M-H2O+H]+ TRUE C44H84NO8P 1120 Perillic acid 9,62 167,10662 166,09934 [M+H]+ TRUE C10H14O2 2110 Phenylacetaldehyde 12,86 121,06528 120,05801 [M+H]+ TRUE C8H8O 2120 Phenylpropanolamine 8,45 152,10621 151,09894 [M+H]+ TRUE C9H13NO 2110 Phosphoric acid 1,18 98,98442 97,97714 [M+H]+ TRUE H3O4P 2200 Picolinic acid 1,76 124,03928 123,032 [M+H]+ TRUE C6H5NO2 2100 Pregnenolone 18,08 317,24375 316,23647 [M+H]+ TRUE C21H32O2 1110 Propanal 0,81 59,04942 58,04214 [M+H]+ TRUE C3H6O 2120 Protocatechuic acid 1,84 177,01952 154,03006 [M+Na]+, [M-H2O+H]+, [M+H]+ TRUE C7H6O4 1110 Pyridine 3,16 80,04931 79,04203 [M+H]+ TRUE C5H5N 2220 Pyridoxal 5'-phosphate 2,81 248,03475 247,02747 [M+H]+ TRUE C8H10NO6P 1110 Pyroglutamic acid 6,92 130,04952 129,04224 [M+H]+ TRUE C5H7NO3 2120 Quinaldic acid 6,61 174,0547 173,04742 [M+H]+ TRUE C10H7NO2 2220 Salbutamol 7,46 240,15607 239,14879 [M+H]+ TRUE C13H21NO3 1110 Sphinganine 12,91 302,30635 301,29907 [M+H]+ TRUE C18H39NO2 2110 Sphingosine 12,61 282,2824 299,28446 [M-H2O+H]+, [M+H]+ TRUE C18H37NO2 1110 Succinic acid 2,42 119,03507 118,0278 [M+H]+ TRUE C4H6O4 2110 Tetracosanoic acid 17,88 369,37319 368,36591 [M+H]+ TRUE C24H48O2 2020 Threonic acid 7,06 137,0447 136,03742 [M+H]+ TRUE C4H8O5 2120 Tolnaftate 8,62 308,11513 307,10785 [M+H]+ TRUE C19H17NOS 1100 Trans-Aconitic acid 22,06 175,02207 174,0148 [M+H]+, [M+H]+ TRUE C6H6O6 2100 Traumatic acid 9,29 229,1423 228,13502 [M+H]+ TRUE C12H20O4 2120 Trimethylamine 1,07 60,081 59,07372 [M+H]+ TRUE C3H9N 2120 Tryptamine 16,03 161,10784 160,10056 [M+H]+ TRUE C10H12N2 2120 Umbelliferone 11,57 163,03476 162,02748 [M+H]+ TRUE C9H6O3 1110 Uracil 5,34 113,034 112,02672 [M+H]+ TRUE C4H4N2O2 2010 Ureidosuccinic acid 13,31 177,05518 176,04791 [M+H]+ TRUE C5H8N2O5 1110 Uridine 5,34 245,07608 244,06876 [M+H]+, [M+Na]+, [M+H]+ TRUE C9H12N2O6 2020 Uridine 5'-monophosphate 2,22 325,04371 324,03774 [M+H]+, [M+Na]+ TRUE C9H13N2O9P 2210 Uridine diphosphate-N-acetylglucosamine 1,82 608,09215 607,08406 [M+H]+, [M+Na]+ TRUE C17H27N3O17P2 1220 Urocanic acid 2,64 139,04934 138,04206 [M+H]+ TRUE C6H6N2O2 2120 METABOLITES_END #END