#METABOLOMICS WORKBENCH SIMR_Core_Facility_20230504_011611 DATATRACK_ID:4011 STUDY_ID:ST002703 ANALYSIS_ID:AN004383 PROJECT_ID:PR001674 VERSION 1 CREATED_ON May 7, 2023, 11:15 pm #PROJECT PR:PROJECT_TITLE Multi-Omics Analysis Revealed a Significant Molecular Changes in PR:PROJECT_TITLE Doxorubicin-Resistant Lung Cancer Cells. PR:PROJECT_TYPE LC-MS/MS PR:PROJECT_SUMMARY Lung cancer is the second most common cancer and the leading cause of PR:PROJECT_SUMMARY cancer-related deaths worldwide. Chemotherapy resistance in lung cancer is one PR:PROJECT_SUMMARY of the major characteristics of an aggressive phenotype with poor prognosis. PR:PROJECT_SUMMARY Therefore, there is a critical need to explore the significant molecular changes PR:PROJECT_SUMMARY associated with resistance to conventional chemotherapy, and identify potential PR:PROJECT_SUMMARY therapeutic targets for treatment of resistant lung cancer. In this study, we PR:PROJECT_SUMMARY have performed comprehensive quantitative proteomics and metabolomics analysis PR:PROJECT_SUMMARY of non-small cell lung cancer cells (A549-P) and doxorubicin resistant A549 PR:PROJECT_SUMMARY cells (A549-R), using state-of-the-art Trapped Ion Mobility Spectroscopy, PR:PROJECT_SUMMARY Time-of-Flight Mass Spectrometry (TIMS-TOF-MS). The results revealed 30 PR:PROJECT_SUMMARY dysregulated proteins and 37 significantly altered metabolites in A549-R cells PR:PROJECT_SUMMARY compared to A549-P cells. Among the significantly upregulated proteins are liver PR:PROJECT_SUMMARY carboxylesterase 1, anterior gradient protein 2 homolog and nicotinamide PR:PROJECT_SUMMARY phosphoribosyltransferase. A group of the upregulated proteins are endogenous PR:PROJECT_SUMMARY and xenobiotic-metabolizing enzymes, including UDP-glucuronosyltransferase 1-6, PR:PROJECT_SUMMARY CES1, and epoxide hydrolase 1. While Importin, ATP-citrate synthase and CTP PR:PROJECT_SUMMARY synthase are downregulated. The significantly altered metabolites include PR:PROJECT_SUMMARY sepiapterin, glutathione, glycine, pyridine and niacinamide. The performed PR:PROJECT_SUMMARY multi-omics integrated analysis revealed the involvement of purine and PR:PROJECT_SUMMARY glutathione metabolism, ABC transporters, citric acid cycle in the development PR:PROJECT_SUMMARY of resistance in A549 cells, besides the involvement of energy metabolism, PR:PROJECT_SUMMARY pathways related to cancer progression, invasion and migration, and redox PR:PROJECT_SUMMARY homeostasis. Collectively, this exploratory study effectively revealed the PR:PROJECT_SUMMARY significantly dysregulated proteins and metabolites in doxorubicin resistant PR:PROJECT_SUMMARY A549 cells and shed the light on potential biomarkers for chemotherapy resistant PR:PROJECT_SUMMARY non-small cell lung cancer. In addition, multi-omics integrated analysis PR:PROJECT_SUMMARY elucidates the involved pathways in resistance including pathways related to PR:PROJECT_SUMMARY progression and invasion which would improve prognosis and open the door for new PR:PROJECT_SUMMARY potential therapeutic targets. PR:INSTITUTE Sharjah Institute for Medical Research PR:DEPARTMENT Research institute of medical and health science PR:LABORATORY Biomarker Discovery Group 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 Multi-Omics Analysis Revealed a Significant Molecular Changes in ST:STUDY_TITLE Doxorubicin-Resistant Lung Cancer Cells. ST:STUDY_TYPE LC/MS/MS ST:STUDY_SUMMARY Lung cancer is the second most common cancer and the leading cause of ST:STUDY_SUMMARY cancer-related deaths worldwide. Chemotherapy resistance in lung cancer is one ST:STUDY_SUMMARY of the major characteristics of an aggressive phenotype with poor prognosis. ST:STUDY_SUMMARY Therefore, there is a critical need to explore the significant molecular changes ST:STUDY_SUMMARY associated with resistance to conventional chemotherapy, and identify potential ST:STUDY_SUMMARY therapeutic targets for treatment of resistant lung cancer. In this study, we ST:STUDY_SUMMARY have performed comprehensive quantitative proteomics and metabolomics analysis ST:STUDY_SUMMARY of non-small cell lung cancer cells (A549-P) and doxorubicin resistant A549 ST:STUDY_SUMMARY cells (A549-R), using state-of-the-art Trapped Ion Mobility Spectroscopy, ST:STUDY_SUMMARY Time-of-Flight Mass Spectrometry (TIMS-TOF-MS). The results revealed 30 ST:STUDY_SUMMARY dysregulated proteins and 37 significantly altered metabolites in A549-R cells ST:STUDY_SUMMARY compared to A549-P cells. Among the significantly upregulated proteins are liver ST:STUDY_SUMMARY carboxylesterase 1, anterior gradient protein 2 homolog and nicotinamide ST:STUDY_SUMMARY phosphoribosyltransferase. A group of the upregulated proteins are endogenous ST:STUDY_SUMMARY and xenobiotic-metabolizing enzymes, including UDP-glucuronosyltransferase 1-6, ST:STUDY_SUMMARY CES1, and epoxide hydrolase 1. While Importin, ATP-citrate synthase and CTP ST:STUDY_SUMMARY synthase are downregulated. The significantly altered metabolites include ST:STUDY_SUMMARY sepiapterin, glutathione, glycine, pyridine and niacinamide. The performed ST:STUDY_SUMMARY multi-omics integrated analysis revealed the involvement of purine and ST:STUDY_SUMMARY glutathione metabolism, ABC transporters, citric acid cycle in the development ST:STUDY_SUMMARY of resistance in A549 cells, besides the involvement of energy metabolism, ST:STUDY_SUMMARY pathways related to cancer progression, invasion and migration, and redox ST:STUDY_SUMMARY homeostasis. Collectively, this exploratory study effectively revealed the ST:STUDY_SUMMARY significantly dysregulated proteins and metabolites in doxorubicin resistant ST:STUDY_SUMMARY A549 cells and shed the light on potential biomarkers for chemotherapy resistant ST:STUDY_SUMMARY non-small cell lung cancer. In addition, multi-omics integrated analysis ST:STUDY_SUMMARY elucidates the involved pathways in resistance including pathways related to ST:STUDY_SUMMARY progression and invasion which would improve prognosis and open the door for new ST:STUDY_SUMMARY potential therapeutic targets. ST:INSTITUTE University of Sharjah ST:DEPARTMENT Research institute of medical and health science ST:LABORATORY Biomarker Discovery Group 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 #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 - P Control 1A-01_7_1_3213 Treatment:A549-P RAW_FILE_NAME=P Control 1A-01_7_1_3213.d SUBJECT_SAMPLE_FACTORS - P Control 1A-02_7_1_3214 Treatment:A549-P RAW_FILE_NAME=P Control 1A-02_7_1_3214.d SUBJECT_SAMPLE_FACTORS - P Control 2A-01_8_1_3215 Treatment:A549-P RAW_FILE_NAME=P Control 2A-01_8_1_3215.d SUBJECT_SAMPLE_FACTORS - P Control 2A-02_8_1_3216 Treatment:A549-P RAW_FILE_NAME=P Control 2A-02_8_1_3216.d SUBJECT_SAMPLE_FACTORS - P Control 3A-01_9_1_3217 Treatment:A549-P RAW_FILE_NAME=P Control 3A-01_9_1_3217.d SUBJECT_SAMPLE_FACTORS - P Control 3A-02_9_1_3222 Treatment:A549-P RAW_FILE_NAME=P Control 3A-02_9_1_3222.d SUBJECT_SAMPLE_FACTORS - R Control 1A-01_13_1_3229 Treatment:A549-P RAW_FILE_NAME=R Control 1A-01_13_1_3229.d SUBJECT_SAMPLE_FACTORS - R Control 1A-02_13_1_3230 Treatment:A549-R RAW_FILE_NAME=R Control 1A-02_13_1_3230.d SUBJECT_SAMPLE_FACTORS - R Control 2A-01_14_1_3231 Treatment:A549-R RAW_FILE_NAME=R Control 2A-01_14_1_3231.d SUBJECT_SAMPLE_FACTORS - R Control 2A-02_14_1_3232 Treatment:A549-R RAW_FILE_NAME=R Control 2A-02_14_1_3232.d SUBJECT_SAMPLE_FACTORS - R Control 3A-01_15_1_3233 Treatment:A549-R RAW_FILE_NAME=R Control 3A-01_15_1_3233.d SUBJECT_SAMPLE_FACTORS - R Control 3A-02_15_1_3234 Treatment:A549-R RAW_FILE_NAME=R Control 3A-02_15_1_3234.d #COLLECTION CO:COLLECTION_SUMMARY The human non-small cell lung cancer cell line (A549) utilized in this study was CO:COLLECTION_SUMMARY purchased from AmericanType Culture Collection (ATCC) (Manassas, USA). A CO:COLLECTION_SUMMARY doxorubicin-resistant A549 cell line was generated in our laboratory (Drug CO:COLLECTION_SUMMARY Design and Discovery Research Group, Pharmacology Laboratory, Sharjah Institute CO:COLLECTION_SUMMARY for Medical Research, University of Sharjah). Doxorubicin (Dox) and irinotecan CO:COLLECTION_SUMMARY were purchased from Sigma-Aldrich (Darmstadt, Germany) and Abcam (Cambridge, CO:COLLECTION_SUMMARY UK), respectively. A Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 CO:COLLECTION_SUMMARY (1:1) (DMEM/F-12) medium was purchased from Gibco, Thermo Fisher Scientific CO:COLLECTION_SUMMARY (Massachusetts, USA). Fetal bovine serum (FBS), penicillin/streptomycin, CO:COLLECTION_SUMMARY trypsin, and trypan blue were purchased from Sigma-Aldrich (Darmstadt, Germany). CO:COLLECTION_SUMMARY For cell viability assay: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl CO:COLLECTION_SUMMARY tetrazolium bromide) reagent and dimethyl sulfoxide (DMSO) were purchased from CO:COLLECTION_SUMMARY Sigma-Aldrich (Darmstadt, Germany). Protease inhibitor cocktail tablets were CO:COLLECTION_SUMMARY purchased from Roche (Mannheim, Germany). Methanol (≥99.9%), acetonitrile CO:COLLECTION_SUMMARY (ACN) and deionized water, as well as LC-MS CHROMASOLV, were purchased from CO:COLLECTION_SUMMARY Honeywell (Seelze, Germany). Trifluoroacetic acid (TFA) was purchased from CO:COLLECTION_SUMMARY Sigma-Aldrich (Darmstadt, Germany) and acetonitrile (ACN) was purchased from CO:COLLECTION_SUMMARY Honeywell (Charlotte, USA). Lys-C endopeptidase was purchased from Fujifilm Wako CO:COLLECTION_SUMMARY Chemicals (Virginia, USA). For western blot analysis: primary antibodies against CO:COLLECTION_SUMMARY liver carboxylesterase 1/CES1 and β-actin were obtained from Abcam (Cambridge, CO:COLLECTION_SUMMARY UK) and Cell Signaling Technologies (Danvers, USA), respectively. Anti-rabbit CO:COLLECTION_SUMMARY IgG, Horseradish Peroxidase (HRP)-linked secondary antibody was purchased from CO:COLLECTION_SUMMARY Cell Signaling Technologies (Danvers, USA). Skimmed milk was purchased from CO:COLLECTION_SUMMARY Sigma-Aldrich (Darmstadt, Germany). For Enzyme-linked immunosorbent assay CO:COLLECTION_SUMMARY (ELISA): carboxylesterase 1 (CES1) specific activity assay kit purchased from CO:COLLECTION_SUMMARY Abcam (Cambridge, UK). CO:SAMPLE_TYPE Lung #TREATMENT TR:TREATMENT_SUMMARY The human non-small cell lung cancer cell line (A549) was purchased from TR:TREATMENT_SUMMARY AmericanType Culture Collection (ATCC) (Manassas, USA). A doxorubicin-resistant TR:TREATMENT_SUMMARY A549 (A549-R) cell line was generated in our laboratory (Drug Design and TR:TREATMENT_SUMMARY Discovery Research Group, Pharmacology Laboratory, Sharjah Institute for Medical TR:TREATMENT_SUMMARY Research, University of Sharjah). To develop resistant cells from the parental TR:TREATMENT_SUMMARY A549 cells (A549-P), A549-P cells were treated with a tenth of Dox's maximal TR:TREATMENT_SUMMARY inhibitory concentration (IC10). The surviving population of cells was cultured TR:TREATMENT_SUMMARY in a new flask and treated with a gradual increase of Dox concentration for 8 TR:TREATMENT_SUMMARY months. Maintenance of A549-R cells resistance was at a dose of 0.02 µg/ml of TR:TREATMENT_SUMMARY Dox. A549-P and A549-R cells were cultured in a Dulbecco's Modified Eagle TR:TREATMENT_SUMMARY Medium/ Nutrient Mixture F-12 (1:1) medium (DMEM/F-12) supplemented with 10% TR:TREATMENT_SUMMARY fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were TR:TREATMENT_SUMMARY incubated in a humidified atmosphere of 5% CO2 at 37 ˚C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Two million cells from both cell lines (A549-P and A549-R) were seeded in T75 SP:SAMPLEPREP_SUMMARY cell culture flasks. Following culturing the cells, they were collected by SP:SAMPLEPREP_SUMMARY trypsin and a pellet of 3x106 cells was stored at -80 ℃ for further analysis. SP:SAMPLEPREP_SUMMARY Each cell line was prepared in triplicates for the metabolomics and proteomics SP:SAMPLEPREP_SUMMARY studies. Frozen A549-P and A549-R cells pellets were centrifuged at 14000 rpm SP:SAMPLEPREP_SUMMARY for 5 min at 4 ℃ to separate them from Phosphate Buffered Saline (PBS) buffer. SP:SAMPLEPREP_SUMMARY The pellets were suspended in a lysis buffer (10 mM Tris) and protease inhibitor SP:SAMPLEPREP_SUMMARY cocktail tablets. The lysed cells were kept on ice for 10 min, and then each SP:SAMPLEPREP_SUMMARY sample was vortexed for 30 s. After that, the samples were sonicated in an ice SP:SAMPLEPREP_SUMMARY bath using a Q500 sonicator from QSonica Sonicator (Fairfield, Connecticut, USA) SP:SAMPLEPREP_SUMMARY at 30% amp for 30 s. Then, the samples were centrifuged at 14000 rpm for 5 min SP:SAMPLEPREP_SUMMARY at 4℃, 400 µL of methanol was added to the supernatant, followed by 300 µL SP:SAMPLEPREP_SUMMARY of chloroform and vortexed for 30 seconds, and then centrifuged at 14000 rpm for SP:SAMPLEPREP_SUMMARY 5 min at 4 ℃. The resulting aqueous biphasic solution's upper layer was SP:SAMPLEPREP_SUMMARY transferred carefully into LC vials without touching the white disk for SP:SAMPLEPREP_SUMMARY metabolites analysis. For proteins extraction, the generated white disk and the SP:SAMPLEPREP_SUMMARY lower layer were washed with 300 µL of methanol and vortexed vigorously, and SP:SAMPLEPREP_SUMMARY then centrifuged at 14000 rpm for 3 min at 4 ℃. After that, the protein pellet SP:SAMPLEPREP_SUMMARY was precipitated and allowed to dry by keeping the tubes opened for 5 min. Then SP:SAMPLEPREP_SUMMARY a denaturation buffer was used to re-suspend the pellets (10 mM Tris, 6 M Urea, SP:SAMPLEPREP_SUMMARY 2 M Thiourea, pH 8) and the concentration of precipitated proteins was SP:SAMPLEPREP_SUMMARY determined using the modified Bradford assay. The resulted supernatant from the SP:SAMPLEPREP_SUMMARY previous centrifugation, which contains metabolites, was transferred to the SP:SAMPLEPREP_SUMMARY previously collected metabolites in the LC vials. The metabolites samples were SP:SAMPLEPREP_SUMMARY dried at 45 ℃ using an EZ-2 Plus evaporator from GeneVac (Ipswich, UK), and SP:SAMPLEPREP_SUMMARY then the dehydrated extracts were reconstituted in 200 µL 0.1% formic acid (FA) SP:SAMPLEPREP_SUMMARY in water and vortexed for 2 min. Finally, the samples were filtered using a SP:SAMPLEPREP_SUMMARY hydrophilic syringe filter with a pore size of 0.45 µm and were ready to be SP:SAMPLEPREP_SUMMARY analyzed using Q-TOF MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Bruker timsTOF CH:COLUMN_NAME Hamilton Intensity Solo 2 C18 CH:SOLVENT_A Water (0.1% Formic Acid) CH:SOLVENT_B ACN (0.1% Formic Acid) CH:FLOW_GRADIENT 1% B was held for 2 min, ramping to 99% B over 15 min, and held at 99% B for 3 CH:FLOW_GRADIENT min before re-equilibrating to 1% B for 10 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 MetaboScape® 4.0 software from Bruker (Bremen, Germany) was used for data MS:MS_COMMENTS processing. The parameters for the detection were as follows: a minimum of 7 MS:MS_COMMENTS spectra were used for peak detection, while for molecular features detection, MS:MS_COMMENTS the minimum intensity threshold was equivalent to 1000 counts. Mass MS:MS_COMMENTS recalibration was performed within a retention time range between 0-0.3 min. MS:MS_COMMENTS Only those features present in at least 2 of 6 samples were considered. Finally, MS:MS_COMMENTS the detected MS/MS spectra assigned onto the bucket table allowed for better MS:MS_COMMENTS viewing and understanding. The main features included retention time, measured MS:MS_COMMENTS m/z, detected fragments, and molecular weight [12]. Data bucketing parameters MS:MS_COMMENTS were as follows: the retention time range was between 0.3 min and 25 min, MS:MS_COMMENTS whereas the mass range began at 50 m/z and finished at 1,300 m/z. A two-tailed MS:MS_COMMENTS independent student’s t-test was utilized to determine the significantly MS:MS_COMMENTS altered metabolites between the two cell lines. The threshold for significance MS:MS_COMMENTS was p-value <0.05. Functional enrichment analysis was constructed utilizing the MS:MS_COMMENTS MetaboAnalyst 5.0 website (https://www.metaboanalyst.ca). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples P Control 1A-01_7_1_3213 P Control 1A-02_7_1_3214 P Control 2A-01_8_1_3215 P Control 2A-02_8_1_3216 P Control 3A-01_9_1_3217 P Control 3A-02_9_1_3222 R Control 1A-01_13_1_3229 R Control 1A-02_13_1_3230 R Control 2A-01_14_1_3231 R Control 2A-02_14_1_3232 R Control 3A-01_15_1_3233 R Control 3A-02_15_1_3234 Factors Treatment:A549-P Treatment:A549-P Treatment:A549-P Treatment:A549-P Treatment:A549-P Treatment:A549-P Treatment:A549-P Treatment:A549-R Treatment:A549-R Treatment:A549-R Treatment:A549-R Treatment:A549-R 1,3-Diaminopropane 4666 4950 4736 5452 4494 4692 4916 3660 4642 4304 3980 4188 2-Hydroxycaproic acid 129750 133600 132376 136624 139962 141430 124708 73172 152862 134634 132716 117550 2-Phenylbutyric acid 27492 27162 25808 20876 28068 26324 27792 26216 27266 25844 26086 26520 2-Pyrrolidinone 40206 47180 37432 32900 44966 52928 39438 50688 45322 60390 46074 48544 3,4-Dihydroxyphenylglycol 49352 49876 39154 53566 51466 51458 47496 51390 35986 67926 58596 28532 3-Aminoisobutanoic acid 4542 7904 9274 10314 12070 7360 11694 10898 8554 8844 11636 9488 3-Hexenedioic acid 2758 2096 2636 2594 2012 3024 1216 1494 1966 1822 1566 1702 4-Deoxytetronic acid 3176 2696 2590 2742 2402 3348 3376 2530 2540 2650 2976 3244 5'-Methylthioadenosine 89326 93880 80372 78822 89568 93240 109102 85996 120472 133722 141504 139398 5-Methoxytryptophan 6070 4260 4664 4758 4218 4852 4486 5016 4894 3990 5956 4998 Acrylamide 338974 369194 559616 624776 412038 389466 342106 355470 404884 316514 663176 639770 Adenine 13482 12038 16304 13024 15532 12770 10688 12540 13592 11460 13146 10768 Adenosine monophosphate 126486 113730 129910 114952 110114 165482 130388 105730 137046 186344 126666 146450 Adipic acid 2706 2770 2790 2826 2808 2648 2444 2822 2942 2706 2566 2594 Androsterone 30372 27994 32382 32804 30134 29970 29754 31086 30876 30876 31460 28412 Aspartame 2870 2454 2454 2648 2838 2988 2810 3452 3238 3462 3388 3292 Benzaldehyde 15134 13826 19738 18778 14126 13728 15208 14904 10504 13334 18804 15988 Benzoic acid 4222 2434 2170 1838 1890 1802 1370 1706 1850 1998 3356 3084 Cadaverine 16576 28840 33934 34366 36740 33344 35096 31846 40024 37444 38796 38850 Cinnamic acid 3040 2758 3592 3210 4494 4132 4096 2694 4272 3610 2762 2896 Deoxyguanosine 55604 52110 59818 55324 61244 58054 50176 43914 48436 47030 48688 53178 Diacetyl 25748 26650 26334 24290 26642 25554 26404 27456 25326 26162 27436 28416 Elaidic acid 34676 18278 17600 15184 14446 9968 5498 4906 4196 3794 4362 3782 Glutathione 2782 2770 836 858 3396 5400 11296 9338 13408 18076 9626 9442 Glycerol 44590 44698 52322 49694 58240 60078 59504 51814 66606 52126 53682 59342 Glycerophosphocholine 3084 2986 3258 2986 3354 3360 1138 0 1176 1232 1184 1020 Glycine 4732 2594 982 1532 4718 1714 18784 17200 17210 3488 6218 4350 Guanine 69332 69710 78770 76330 88258 79136 18018 19610 14222 13778 19104 18596 Guanosine 43690 41010 44648 48798 50776 52374 9654 9172 7736 7698 8698 9386 Guanosine 5'-diphosphate 0 1698 1624 0 0 0 2440 2510 3364 3634 3078 2642 Guanosine monophosphate 19882 18176 21484 16560 18382 30762 14850 13908 15262 23868 18252 19214 Hydroxyisocaproic acid 7212 5252 6102 6088 6822 7576 6706 5848 6812 5942 7504 8310 Hyodeoxycholic acid 29802 22974 24852 20506 15484 11978 2536 2330 1956 1852 2362 2088 Hypoxanthine 414936 409348 488478 479712 476228 471068 247148 232802 236784 233282 266476 241672 Indole 5306 6600 5578 4756 5462 5824 4042 4986 5902 5572 6088 6046 Indoleacetic acid 8782 7496 7310 5548 8856 7954 3874 2936 3234 4328 3300 2114 Indoleacrylic acid 107972 102312 94114 88078 100024 106136 82120 85466 83794 96490 89766 87994 Inosine 89586 85442 97972 96952 114178 96974 49736 44548 49518 50598 53560 52226 Inosinic acid 7490 2880 3146 2110 2922 3946 2548 0 2854 3896 2860 3296 Isobutyric acid 117868 111066 126220 122226 108202 126584 125242 129748 117574 118116 92666 130906 L-Acetylcarnitine 49830 52108 50836 46274 48458 72428 30994 25718 33570 44148 28058 35720 L-Arginine 1808 2130 2310 2178 3556 2618 1676 1486 1714 2442 2776 2478 L-Carnitine 4072 4164 4090 3848 3654 4206 2098 0 2494 2470 2084 1894 L-Glutamic acid 5306 4506 4726 3416 4950 3754 1468 1542 1656 1740 1684 0 L-Norleucine 14210 10248 11996 10356 10284 11644 14146 11524 10250 10446 11862 11292 L-Phenylalanine 40972 40338 38690 38824 48862 43682 37324 39022 49920 41022 36470 34364 L-Proline 2758 2776 3376 2676 3150 2242 3160 2900 2918 2988 2532 3470 L-Tryptophan 20892 21290 19468 20268 23762 23194 16866 17464 18516 21698 22712 19718 m-Coumaric acid 4948 4804 5352 3810 4996 5406 5616 2844 3918 6354 5992 5630 Maltitol 17972 17278 17378 18740 18680 16402 19414 17808 17892 17520 18180 17202 Melatonin 3472 3800 1470 2586 3082 2934 2658 2556 3320 4054 6012 6326 Mevalonic acid 0 4946 5332 4698 4456 5374 5706 4252 4750 6310 4836 5668 N,N-Dimethylformamide 44134 42010 85880 123976 95296 48984 43942 43670 64074 31606 0 71886 N-Acetyl-L-alanine 7182 7548 7708 7312 8784 5886 10010 8064 8364 8170 8366 8530 N-Acetylputrescine 6920 6862 7638 8072 6972 7896 6650 7212 8640 8702 9478 9600 N-Acetylserotonin 15098 15134 13978 13402 7016 14418 14000 14388 12646 13492 14548 14632 Neopterin 34716 32648 25976 26796 37730 40818 27448 22074 53484 45790 56732 61964 Niacinamide 44204 40882 44918 38278 47766 60762 85702 86176 100798 117980 76038 88232 Nutriacholic acid 196888 196570 209052 184896 184298 124680 17778 18148 14768 14686 10918 8368 o-Tyrosine 5476 6402 5108 5830 7848 4192 5382 5660 6034 4682 5136 3818 PA(16:0/16:0) 3774 3998 2808 4714 2388 4904 1268 1224 2118 3096 2110 2532 Pantothenic acid 87140 88444 82496 82318 91272 90878 81310 76630 94748 98310 88456 89298 PC(16:0/16:0) 0 0 0 0 0 0 8602 8650 7440 6874 6754 7784 PC(18:1(9Z)/18:1(9Z)) 0 0 0 0 0 0 17448 15382 12328 8250 4750 9668 Phenylacetaldehyde 35290 34058 31054 30522 33188 34150 31338 35536 34672 32940 31760 30484 Phenylacetylglycine 9318 8522 9368 9380 8220 9714 11042 8232 9430 7664 8528 8520 Propionic acid 6210 6144 5924 7122 6956 5114 5770 6558 7854 6448 8218 7072 Pyridine 16586 15978 18936 16222 20662 15794 36132 35030 38042 41546 32354 40930 Pyridoxal 5804 3012 2274 2320 2524 1980 2494 2792 2284 2136 2848 2214 Pyroglutamic acid 5316 5790 5112 5214 6948 7342 4788 3452 5616 6672 7568 6198 Rhamnose 3272 2736 3086 3136 3066 3102 3882 2966 3512 3440 3586 3246 S-Adenosylhomocysteine 14544 12030 16226 17718 19518 2272 19074 16568 26044 4276 12808 8596 Saccharopine 4870 6466 8142 8542 7304 4996 11492 12758 8622 10204 9050 11448 Sepiapterin 492 748 0 274 482 610 2414 2148 2704 2662 3646 3714 Sorbitol 19698 17646 20920 17134 19744 17832 20806 20848 22738 21440 19542 21106 Spermine 18312 17812 18844 20688 20674 21266 16026 15790 17732 18086 16560 15504 Sphingosine 47958 44864 56882 49512 48900 37086 24178 30206 37650 29644 27922 30272 Succinic acid 3102 2532 3324 3578 3214 4214 1494 430 1778 1964 1720 1712 Succinylacetone 29980 12674 11440 8468 4616 12536 656 1704 1674 1752 1964 1478 Tartaric acid 5024 4580 5344 4580 4798 7460 5036 4206 5012 6674 5604 6106 Terephthalic acid 25212 19066 19482 16254 16784 15156 2636 2982 2648 2600 2316 2190 Threonic acid 53502 56334 47724 36850 35132 81322 29788 28934 27554 36682 32086 36372 Uric acid 36826 37622 32094 34220 42244 45280 32488 30258 51884 51814 52112 51528 Xanthurenic acid 233666 221774 239682 227832 224196 226124 229436 230114 223512 204408 78672 168300 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT [min] m/z meas. M meas. Ions MS/MS Molecular Formula 1,3-Diaminopropane 2.14 75.09174 74.08446 [M+H]+ TRUE C3H10N2 2-Hydroxycaproic acid 16.29 133.08644 132.07916 [M+H]+ TRUE C6H12O3 2-Phenylbutyric acid 17.42 165.09137 164.08409 [M+H]+ TRUE C10H12O2 2-Pyrrolidinone 4.82 86.06006 85.05279 [M+H]+ TRUE C4H7NO 3,4-Dihydroxyphenylglycol 3.82 171.06273 170.05545 [M+H]+ TRUE C8H10O4 3-Aminoisobutanoic acid 15.39 104.07088 103.0636 [M+H]+ TRUE C4H9NO2 3-Hexenedioic acid 3.49 145.04942 144.04215 [M+H]+ TRUE C6H8O4 4-Deoxytetronic acid 3.86 87.04424 86.03696 [M+H]+ TRUE C4H6O2 5'-Methylthioadenosine 7.38 298.09649 297.08921 [M+H]+ TRUE C11H15N5O3S 5-Methoxytryptophan 7.17 235.10531 234.09803 [M+H]+ TRUE C12H14N2O3 Acrylamide 8.14 72.04421 71.03694 [M+H]+ TRUE C3H5NO Adenine 6.18 136.06178 135.0545 [M+H]+ TRUE C5H5N5 Adenosine monophosphate 3.56 348.07057 347.06329 [M+H]+ TRUE C10H14N5O7P Adipic acid 3.36 147.06523 146.05796 [M+H]+ TRUE C6H10O4 Androsterone 17.35 291.23245 290.22518 [M+H]+ TRUE C19H30O2 Aspartame 6.81 295.12801 294.12073 [M+H]+ TRUE C14H18N2O5 Benzaldehyde 13.95 107.04938 106.0421 [M+H]+ TRUE C7H6O Benzoic acid 14.28 123.04638 122.03911 [M+H]+ TRUE C7H6O2 Cadaverine 15.05 103.12326 102.11599 [M+H]+ TRUE C5H14N2 Cinnamic acid 6.71 149.05979 148.05252 [M+H]+ TRUE C9H8O2 Deoxyguanosine 6.18 268.10415 267.09687 [M+H]+ TRUE C10H13N5O4 Diacetyl 16.33 87.04393 86.03665 [M+H]+ TRUE C4H6O2 Elaidic acid 19.58 283.2638 282.25655 [M+H]+, [M-H2O+H]+ TRUE C18H34O2 Glutathione 3.45 308.09126 307.08399 [M+H]+ TRUE C10H17N3O6S Glycerol 3.22 93.05484 92.04757 [M+H]+ TRUE C3H8O3 Glycerophosphocholine 3.29 258.11024 257.10296 [M+H]+ TRUE C8H20NO6P Glycine 5.96 76.03957 75.0323 [M+H]+ TRUE C2H5NO2 Guanine 6.34 152.05677 151.04949 [M+H]+ TRUE C5H5N5O Guanosine 6.34 284.09914 283.09186 [M+H]+ TRUE C10H13N5O5 Guanosine 5'-diphosphate 6.2 444.03225 443.02497 [M+H]+ TRUE C10H15N5O11P2 Guanosine monophosphate 3.71 364.06563 363.05835 [M+H]+ TRUE C10H14N5O8P Hydroxyisocaproic acid 7.02 133.08583 132.07856 [M+H]+ TRUE C6H12O3 Hyodeoxycholic acid 21.28 393.29772 392.29045 [M+H]+ TRUE C24H40O4 Hypoxanthine 6.35 137.04569 136.03842 [M+H]+, [M-H2O+H]+ TRUE C5H4N4O Indole 7.54 118.06504 117.05776 [M+H]+ TRUE C8H7N Indoleacetic acid 12.04 176.07087 175.0636 [M+H]+ TRUE C10H9NO2 Indoleacrylic acid 7.54 188.07047 187.06319 [M+H]+ TRUE C11H9NO2 Inosine 6.35 269.08849 268.08122 [M+H]+ TRUE C10H12N4O5 Inosinic acid 3.69 349.05659 348.04932 [M+H]+ TRUE C10H13N4O8P Isobutyric acid 16.34 89.05945 88.05218 [M+H]+ TRUE C4H8O2 L-Acetylcarnitine 3.49 204.12314 203.11586 [M+H]+ TRUE C9H17NO4 L-Arginine 6.25 175.11919 174.11191 [M+H]+ TRUE C6H14N4O2 L-Carnitine 2.93 162.11257 161.10529 [M+H]+ TRUE C7H15NO3 L-Glutamic acid 5.23 148.06043 147.05315 [M+H]+ TRUE C5H9NO4 L-Norleucine 10.94 132.10181 131.09453 [M+H]+ TRUE C6H13NO2 L-Phenylalanine 6.71 166.08626 165.07898 [M+H]+ TRUE C9H11NO2 L-Proline 6.61 116.07088 115.0636 [M+H]+ TRUE C5H9NO2 L-Tryptophan 7.54 205.09704 204.08976 [M+H]+ TRUE C11H12N2O2 m-Coumaric acid 4.71 165.05463 164.04735 [M+H]+ TRUE C9H8O3 Maltitol 3.44 345.13942 344.13214 [M+H]+ TRUE C12H24O11 Melatonin 8.14 233.12634 232.11906 [M+H]+ TRUE C13H16N2O2 Mevalonic acid 3.88 149.08079 148.07351 [M+H]+ TRUE C6H12O4 N,N-Dimethylformamide 5.11 74.06002 73.05274 [M+H]+ TRUE C3H7NO N-Acetyl-L-alanine 3.8 132.06555 131.05827 [M+H]+ TRUE C5H9NO3 N-Acetylputrescine 2.81 131.11786 130.11059 [M+H]+ TRUE C6H14N2O N-Acetylserotonin 7.74 219.11075 218.10347 [M+H]+ TRUE C12H14N2O2 Neopterin 7.68 254.08476 253.07748 [M+H]+ TRUE C9H11N5O4 Niacinamide 3.62 123.05523 122.04796 [M+H]+ TRUE C6H6N2O Nutriacholic acid 21.21 413.26746 390.27829 [M+Na]+, [M+K]+, [M+H]+ TRUE C24H38O4 o-Tyrosine 4.89 182.08137 181.0741 [M+H]+ TRUE C9H11NO3 PA(16:0/16:0) 20.43 649.47771 648.47043 [M+H]+ TRUE C35H69O8P Pantothenic acid 7.1 220.11802 219.11071 [M+H]+, [M+Na]+ TRUE C9H17NO5 PC(16:0/16:0) 14.81 734.56974 733.56247 [M+H]+ TRUE C40H80NO8P PC(18:1(9Z)/18:1(9Z)) 13.72 786.60257 785.59529 [M+H]+ TRUE C44H84NO8P Phenylacetaldehyde 17.42 121.06524 120.05796 [M+H]+ TRUE C8H8O Phenylacetylglycine 6.8 194.07882 193.07155 [M+H]+ TRUE C10H11NO3 Propionic acid 22.76 75.04417 74.03689 [M+H]+ TRUE C3H6O2 Pyridine 3.64 80.04956 79.04228 [M+H]+ TRUE C5H5N Pyridoxal 6.41 168.06194 167.05467 [M+H]+ TRUE C8H9NO3 Pyroglutamic acid 3.86 130.04989 129.04262 [M+H]+ TRUE C5H7NO3 Rhamnose 3.4 165.07583 164.06855 [M+H]+ TRUE C6H12O5 S-Adenosylhomocysteine 5.99 385.12944 384.12209 [M+H]+, [M+H+H]2+ TRUE C14H20N6O5S Saccharopine 1.79 277.13701 276.12973 [M+H]+ TRUE C11H20N2O6 Sepiapterin 6.19 238.08966 237.08238 [M+H]+ TRUE C9H11N5O3 Sorbitol 3.42 183.08636 182.07909 [M+H]+ TRUE C6H14O6 Spermine 2.08 203.22313 202.21585 [M+H]+ TRUE C10H26N4 Sphingosine 13.31 282.27929 299.28266 [M-H2O+H]+, [M+H]+ TRUE C18H37NO2 Succinic acid 3.85 119.03528 118.028 [M+H]+ TRUE C4H6O4 Succinylacetone 20.33 159.0652 158.05793 [M+H]+ TRUE C7H10O4 Tartaric acid 3.99 151.0212 150.01392 [M+H]+ TRUE C4H6O6 Terephthalic acid 21.21 149.02341 166.02671 [M-H2O+H]+, [M+H]+ TRUE C8H6O4 Threonic acid 3.83 137.04562 136.03845 [M+H]+ TRUE C4H8O5 Uric acid 7.5 169.0317 168.02443 [M+H]+ TRUE C5H4N4O3 Xanthurenic acid 8.25 206.04521 205.03793 [M+H]+ TRUE C10H7NO4 METABOLITES_END #END