#METABOLOMICS WORKBENCH SIMR_Core_Facility_20221018_034207 DATATRACK_ID:3519 STUDY_ID:ST002319 ANALYSIS_ID:AN003786 PROJECT_ID:PR001485 VERSION 1 CREATED_ON October 19, 2022, 8:56 am #PROJECT PR:PROJECT_TITLE Mass spectroscopy‑based proteomics and metabolomics analysis of PR:PROJECT_TITLE triple‑positive breast cancer cells treated with tamoxifen and/ or trastuzumab PR:PROJECT_TYPE LC-MS/MS PR:PROJECT_SUMMARY HER2-enriched breast cancer with high levels of hormone receptor expression, PR:PROJECT_SUMMARY known as "triple positive" breast cancer, may represent a new entity with a PR:PROJECT_SUMMARY relatively favourable prognosis against which the combination of chemotherapy, PR:PROJECT_SUMMARY HER-2 inhibition, and endocrine treatment may be considered overtreatment. We PR:PROJECT_SUMMARY explored the effect of the anticancer drugs tamoxifen and trastuzumab, both PR:PROJECT_SUMMARY separately and in combination, on the integrated proteomic and metabolic profile PR:PROJECT_SUMMARY of "triple positive" breast cancer cells (BT-474). Method We employed PR:PROJECT_SUMMARY ultra-high-performance liquid chromatography-quadrupole time of flight mass PR:PROJECT_SUMMARY spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line PR:PROJECT_SUMMARY treated with either tamoxifen, trastuzumab or a combination. Differentially PR:PROJECT_SUMMARY abundant metabolites were identified using the Bruker Human Metabolome Database PR:PROJECT_SUMMARY metabolite library and proteins using the Uniprot proteome for Homo sapiens PR:PROJECT_SUMMARY using MetaboScape and MaxQuant, respectively, for identification and PR:PROJECT_SUMMARY quantitation. Results A total of 77 proteins and 85 metabolites were found to PR:PROJECT_SUMMARY significantly differ in abundance in BT-474 treated cells with tamoxifen 5 PR:PROJECT_SUMMARY μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important PR:PROJECT_SUMMARY cellular signalling pathways which regulate cell growth, apoptosis, PR:PROJECT_SUMMARY proliferation, and chemoresistance, these medicines have a considerable PR:PROJECT_SUMMARY anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include PR:PROJECT_SUMMARY RNA splicing, neutrophil degranulation and activation, cellular redox PR:PROJECT_SUMMARY homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis, PR:PROJECT_SUMMARY ABC transporters and central carbon metabolism. Conclusion Our findings in PR:PROJECT_SUMMARY protein and metabolite level research revealed that anti-cancer drug therapy had PR:PROJECT_SUMMARY a significant impact on the key signalling pathways and molecular processes in PR:PROJECT_SUMMARY triple positive BT-474 cell lines. PR:INSTITUTE Sharjah Institute for Medical Research PR:DEPARTMENT Sharjah Institute for Medical Research 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 Mass spectroscopy‑based proteomics and metabolomics analysis of ST:STUDY_TITLE triple‑positive breast cancer cells treated with tamoxifen and trastuzumab ST:STUDY_SUMMARY HER2-enriched breast cancer with high levels of hormone receptor expression, ST:STUDY_SUMMARY known as "triple positive" breast cancer, may represent a new entity with a ST:STUDY_SUMMARY relatively favourable prognosis against which the combination of chemotherapy, ST:STUDY_SUMMARY HER-2 inhibition, and endocrine treatment may be considered overtreatment. We ST:STUDY_SUMMARY explored the effect of the anticancer drugs tamoxifen and trastuzumab, both ST:STUDY_SUMMARY separately and in combination, on the integrated proteomic and metabolic profile ST:STUDY_SUMMARY of "triple positive" breast cancer cells (BT-474). Method We employed ST:STUDY_SUMMARY ultra-high-performance liquid chromatography-quadrupole time of flight mass ST:STUDY_SUMMARY spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line ST:STUDY_SUMMARY treated with either tamoxifen, trastuzumab or a combination. Differentially ST:STUDY_SUMMARY abundant metabolites were identified using the Bruker Human Metabolome Database ST:STUDY_SUMMARY metabolite library and proteins using the Uniprot proteome for Homo sapiens ST:STUDY_SUMMARY using MetaboScape and MaxQuant, respectively, for identification and ST:STUDY_SUMMARY quantitation. Results A total of 77 proteins and 85 metabolites were found to ST:STUDY_SUMMARY significantly differ in abundance in BT-474 treated cells with tamoxifen 5 ST:STUDY_SUMMARY μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important ST:STUDY_SUMMARY cellular signalling pathways which regulate cell growth, apoptosis, ST:STUDY_SUMMARY proliferation, and chemoresistance, these medicines have a considerable ST:STUDY_SUMMARY anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include ST:STUDY_SUMMARY RNA splicing, neutrophil degranulation and activation, cellular redox ST:STUDY_SUMMARY homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis, ST:STUDY_SUMMARY ABC transporters and central carbon metabolism. Conclusion Our findings in ST:STUDY_SUMMARY protein and metabolite level research revealed that anti-cancer drug therapy had ST:STUDY_SUMMARY a significant impact on the key signalling pathways and molecular processes in ST:STUDY_SUMMARY triple positive BT-474 cell lines. ST:INSTITUTE University of Sharjah ST:DEPARTMENT Sharjah Institute for Medical Research ST:LABORATORY Biomarker Discovery Group ST:LAST_NAME Soares ST:FIRST_NAME Nelson ST:ADDRESS M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah ST:EMAIL nsoares@sharjah.ac.ae ST:PHONE 065057656 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - 1A_93_1_328 Treatment:Control RAW_FILE_NAME=1A''_93_1_328.d SUBJECT_SAMPLE_FACTORS - 1A_93_1_331 Treatment:Control RAW_FILE_NAME=1A''_93_1_331.d SUBJECT_SAMPLE_FACTORS - 1A_92_1_327 Treatment:Control RAW_FILE_NAME=1A'_92_1_327.d SUBJECT_SAMPLE_FACTORS - 1A_92_1_330 Treatment:Control RAW_FILE_NAME=1A'_92_1_330.d SUBJECT_SAMPLE_FACTORS - 1A_91_1_326 Treatment:Control RAW_FILE_NAME=1A_91_1_326.d SUBJECT_SAMPLE_FACTORS - 1A_91_1_329 Treatment:Control RAW_FILE_NAME=1A_91_1_329.d SUBJECT_SAMPLE_FACTORS - 4a_-1_68_1_446 Treatment:COMBINATION RAW_FILE_NAME=4a"_-1_68_1_446.d SUBJECT_SAMPLE_FACTORS - 4a_-2_68_1_447 Treatment:COMBINATION RAW_FILE_NAME=4a"_-2_68_1_447.d SUBJECT_SAMPLE_FACTORS - 4a_-1_67_1_444 Treatment:COMBINATION RAW_FILE_NAME=4a'_-1_67_1_444.d SUBJECT_SAMPLE_FACTORS - 4a_-2_67_1_445 Treatment:COMBINATION RAW_FILE_NAME=4a'_2_67_1_445.d SUBJECT_SAMPLE_FACTORS - 4a-1_60_1_442 Treatment:COMBINATION RAW_FILE_NAME=4a_1_60_1_442.d SUBJECT_SAMPLE_FACTORS - 4a-2_60_1_443 Treatment:COMBINATION RAW_FILE_NAME=4a_2_60_1_443.d #COLLECTION CO:COLLECTION_SUMMARY The BT-474 BC cell line utilized in this study was cultured as monolayers in CO:COLLECTION_SUMMARY DMEM medium supplemented with 10% fetal bovine serum and 1% CO:COLLECTION_SUMMARY penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA). All cultures were CO:COLLECTION_SUMMARY incubated at 37 °C in a humidified atmosphere of 5% CO2. CO:SAMPLE_TYPE Breast cancer cells CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY Triplicate flasks were prepared for each treatment condition for each analysis TR:TREATMENT_SUMMARY (metabolomic and proteomic) for a total of 24 flasks. Two million cells were TR:TREATMENT_SUMMARY seeded in each 75 cm2 tissue culture flask and incubated for 24 h. The cells TR:TREATMENT_SUMMARY were then treated with Tamoxifen (5 μM) and/or Trastuzumab (2.5 μM) for 24 h. TR:TREATMENT_SUMMARY These concentrations correspond to the IC50 of these compounds with BT-474 TR:TREATMENT_SUMMARY cells, as determined by cytotoxicity assays (data not shown). Control cells were TR:TREATMENT_SUMMARY treated with vehicle (dimethyl sulfoxide (DMSO) at 0.5% for 24 h. Following the TR:TREATMENT_SUMMARY incubation period, cells were collected by trypsinization and washed twice with TR:TREATMENT_SUMMARY phosphate-buffered saline solution (PBS) before re-suspending in 1 mL 1 × PBS TR:TREATMENT_SUMMARY for further analysis. Finally, cells were collected as pellets by centrifugation TR:TREATMENT_SUMMARY at 1200 rounds per minute (rpm) for 10 min at room temperature. To negate the TR:TREATMENT_SUMMARY effect of Circadian rhythms on the response of cells to treatment, cells were TR:TREATMENT_SUMMARY kept under the same conditions during the entire incubation period and the cell TR:TREATMENT_SUMMARY collection was done concurrently for all samples. In addition, the same number TR:TREATMENT_SUMMARY of cells were used for each sample to avoid the effect of variation in cell TR:TREATMENT_SUMMARY numbers. TR:TREATMENT Drugs TR:TREATMENT_COMPOUND Tamoxifen and/or Trastuzumab #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sample metabolite extraction A volume of 1 mL of the extraction solvent SP:SAMPLEPREP_SUMMARY (methanol+0.1% formic acid) was added to the cell pellets to quench cells. The SP:SAMPLEPREP_SUMMARY cells were then vortexed for 2 min to ensure the quantitative extraction of the SP:SAMPLEPREP_SUMMARY metabolites and stored on ice for 1 h, during which the samples were vortexed SP:SAMPLEPREP_SUMMARY every 15 min. After this, the insoluble cell matrices were collected and SP:SAMPLEPREP_SUMMARY transferred to centrifuge tubes, intermittent ultrasonication using the COPLEY SP:SAMPLEPREP_SUMMARY sonicator (QSONICA SONICATOR, USA) under 30% amplifier and for 30 s with an ice SP:SAMPLEPREP_SUMMARY bath employed throughout the process. Following that, cells debris were then SP:SAMPLEPREP_SUMMARY centrifuged (15,000 rpm, 10 min, − 4 °C) and the sample supernatants were SP:SAMPLEPREP_SUMMARY collected and transferred to LC vials for drying in the EZ-2 Plus SP:SAMPLEPREP_SUMMARY (GeneVac-Ipswich, UK) at 37±1 °C. Dried samples were resuspended with 200 µL SP:SAMPLEPREP_SUMMARY (water+0.1% formic acid), and vortexed for 2 min. Finally, the samples were SP:SAMPLEPREP_SUMMARY filtered using a hydrophilic Nylon Syringe Filter of 0.45 µm pore size and SP:SAMPLEPREP_SUMMARY analyzed by Q-TOF MS SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were chromatographically separated by inline reversed-phase CH:CHROMATOGRAPHY_SUMMARY chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, CH:CHROMATOGRAPHY_SUMMARY Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B CH:CHROMATOGRAPHY_SUMMARY 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm × 2.1 mm, 1.8 CH:CHROMATOGRAPHY_SUMMARY μm beads) was maintained at 35 ℃ (metabolomics analyses). CH:CHROMATOGRAPHY_TYPE Reversed phase LC CH:INSTRUMENT_NAME Bruker Elute HPG 1300 CH:COLUMN_NAME Hamilton Intensity Solo 2 C18 CH:FLOW_GRADIENT 1%B to 99%B in 15 min CH:FLOW_RATE 250 uL/min CH:COLUMN_TEMPERATURE 35 CH:METHODS_FILENAME . CH:SOLVENT_A Water (0.1% Formic Acid) CH:SOLVENT_B ACN (0.1% Formic Acid) CH:METHODS_ID . CH:COLUMN_PRESSURE . CH:INJECTION_TEMPERATURE . CH:INTERNAL_STANDARD . CH:INTERNAL_STANDARD_MT . CH:RETENTION_INDEX . CH:RETENTION_TIME . CH:SAMPLE_INJECTION . CH:SAMPLING_CONE . CH:ANALYTICAL_TIME . CH:CAPILLARY_VOLTAGE . CH:MIGRATION_TIME . CH:OVEN_TEMPERATURE 35C CH:PRECONDITIONING . CH:RUNNING_BUFFER . CH:RUNNING_VOLTAGE . CH:SHEATH_LIQUID . CH:TIME_PROGRAM . CH:TRANSFERLINE_TEMPERATURE . CH:WASHING_BUFFER . CH:WEAK_WASH_SOLVENT_NAME . CH:WEAK_WASH_VOLUME . CH:STRONG_WASH_SOLVENT_NAME . CH:STRONG_WASH_VOLUME . CH:TARGET_SAMPLE_TEMPERATURE . CH:SAMPLE_LOOP_SIZE . CH:SAMPLE_SYRINGE_SIZE . CH:RANDOMIZATION_ORDER . CH:CHROMATOGRAPHY_COMMENTS . #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 electrosprayionization (ESI) source. The drying gas was set to flow at MS:MS_COMMENTS 10 L/min and the drying temperature to 220℃ and the nebulizer pressure to 2.2 MS:MS_COMMENTS bar. The capillary voltage was 4500 V and the end plate offset 500 V. For MS:MS_COMMENTS metabolomics 20–1300 m/z. The instrument was operated in auto-MS/MS mode. For MS:MS_COMMENTS metabolomics the collision energy was set to 20 eV, the cycle time to 0.5 s with MS:MS_COMMENTS a relative minimum intensity threshold of 400 counts per thousand and a target MS:MS_COMMENTS intensity of 20,000. Sodium formate was injected as an external calibrant in the MS:MS_COMMENTS first 0.3 min of each LC–MS/MS run. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples 1A_93_1_328 1A_93_1_331 1A_92_1_327 1A_92_1_330 1A_91_1_326 1A_91_1_329 4a_-1_68_1_446 4a_-2_68_1_447 4a_-1_67_1_444 4a_-2_67_1_445 4a-1_60_1_442 4a-2_60_1_443 Factors Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:COMBINATION Treatment:COMBINATION Treatment:COMBINATION Treatment:COMBINATION Treatment:COMBINATION Treatment:COMBINATION D-Arginine 1678 2032 1866 1960 2126 2186 948 1202 990 1724 1352 1216 L-Valine 1648 1648 1350 2316 1992 1480 1024 1106 1490 1226 1706 1436 Creatine 2148 2514 3084 2904 2892 3080 110 850 1570 750 900 1302 Trimethylamine 334 130 0 0 0 118 566 1312 782 1132 1222 1342 Cytidine 0 0 0 0 0 0 2136 5390 6336 4942 5180 4796 Adenosine monophosphate 96 112 90 0 540 108 7608 16584 17370 22174 19546 19856 Adenosine 3_,5_-diphosphate 17774 39590 31048 42806 8724 46536 0 230 196 250 270 316 L-Acetylcarnitine 19280 17064 16432 17416 13820 17570 0 0 0 0 0 0 Guanosine monophosphate 4552 7114 7072 7174 20838 6828 0 0 0 60 0 0 Deoxyadenosine monophosphate 1242 1606 1114 1896 0 1944 0 0 0 0 0 0 Pyrrolidonecarboxylic acid 18106 18506 17446 19446 8362 20722 0 0 0 0 0 0 L-Tyrosine 12872 11892 12656 13046 0 13076 0 0 0 100 0 0 Guanosine 0 0 0 0 0 0 0 3904 168 6264 9484 9012 Inosine 14324 11988 12308 11004 572 15786 0 0 0 0 150 0 Metoprolol 308 220 272 0 724 756 6282 6428 8280 6050 5736 5222 L-Fucose 0 0 0 0 0 0 586 1574 1026 1684 1530 1968 Phosphoric acid 0 0 0 0 158 0 1768 1944 2102 2038 2242 2340 Uridine 0 0 0 0 0 0 4506 9742 3720 3814 7082 6956 Phenylacetaldehyde 2254 2582 2434 2752 1974 2418 0 0 86 0 0 0 Aspartyl-lysine 3888 5036 4410 4516 3372 4272 0 0 0 0 0 0 3-Methylindole 0 0 0 0 0 0 896 3424 1370 1398 3866 4188 Linoleic acid 412 332 428 504 1682 672 638 678 1064 1000 946 474 Spermine 43390 42606 37734 42484 47050 36178 250 3732 7578 13208 14892 13568 Glycerophosphocholine 5096 5574 6720 8274 4964 7782 1454 4452 5992 4328 5596 5010 L-Glutamic acid 556 1196 1142 1388 574 1368 82 216 202 94 240 232 Cytidine monophosphate 2464 2586 2556 2718 2094 3350 560 1104 1710 1176 1532 1652 Omeprazole 0 0 0 0 0 0 692 834 1138 1316 1106 1014 Hypoxanthine 0 0 0 0 0 0 6044 15250 8618 0 19310 21942 Uridine diphosphate-N-acetylglucosamine 106 0 0 0 0 0 1192 994 1678 660 1046 1242 Uracil 0 0 0 0 304 0 182 1618 1648 1340 1412 1360 Uridine 5_-monophosphate 3466 4380 4728 4634 4004 5194 0 0 0 0 0 0 Glutathione 472754 387742 463994 435460 469518 487416 102 1122 266 1376 1324 532 L-Leucine 2060 1970 1452 1704 2652 1440 0 196 0 142 374 122 Adenosine 67130 60510 70696 59562 252 111320 96 234 150 0 314 210 L-Phenylalanine 27126 27804 31070 26546 232 30608 0 122 0 174 264 0 5_-Methylthioadenosine 0 0 0 0 0 114 616 1906 1630 1650 2600 2808 L-Tryptophan 98 172 160 0 174 106 4782 12752 10190 16250 14052 14004 2-Ethyl-2-hydroxybutyric acid 1274 1288 1106 1086 1482 908 516 450 362 456 288 382 Isovalerylcarnitine 22672 20998 19624 20818 25046 20210 0 106 0 0 108 138 Sphingosine 17858 14684 16256 13172 11402 15478 0 68 132 164 0 152 PC_18:1_9Z_18:1_9Z_ 0 1604 0 0 0 0 974 1258 4466 2484 9902 7024 PC_16:0_16:0_ 0 0 0 0 0 0 1012 0 17708 2754 14136 8902 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Bucket label RT Formula m/z D-Arginine 174.11176 Da 67.81 s 1.13 C6H14N4O2 175.11903 L-Valine 117.07884 Da 78.21 s 1.3 C5H11NO2 118.08612 Creatine 131.06926 Da 82.43 s 1.37 C4H9N3O2 132.07654 Trimethylamine 59.07385 Da 95.42 s 1.59 C3H9N 60.08113 Cytidine 243.08542 Da 96.12 s 1.6 C9H13N3O5 244.09269 Adenosine monophosphate 347.06332 Da 96.12 s 1.6 C10H14N5O7P 348.07059 Adenosine 3_,5_-diphosphate 427.03111 Da 145.29 s 2.42 C10H15N5O10P2 428.03838 L-Acetylcarnitine 203.11612 Da 147.42 s 2.46 C9H17NO4 204.1234 Guanosine monophosphate 363.05925 Da 149.20 s 2.49 C10H14N5O8P 364.06653 Deoxyadenosine monophosphate 331.06886 Da 151.77 s 2.53 C10H14N5O6P 332.07614 Pyrrolidonecarboxylic acid 129.04249 Da 162.97 s 2.72 C5H7NO3 130.04976 L-Tyrosine 181.07404 Da 233.29 s 3.89 C9H11NO3 182.08132 Guanosine 283.09202 Da 281.30 s 4.69 C10H13N5O5 284.0993 Inosine 268.08141 Da 312.47 s 5.21 C10H12N4O5 269.08869 Metoprolol 267.18408 Da 449.94 s 7.5 C15H25NO3 268.19136 L-Fucose 164.06856 Da 463.12 s 7.72 C6H12O5 165.07583 Phosphoric acid 97.97682 Da 548.95 s 9.15 H3O4P 98.9841 Uridine 244.07157 Da 771.16 s 12.85 C9H12N2O6 245.07885 Phenylacetaldehyde 120.05739 Da 776.02 s 12.93 C8H8O 121.06466 Aspartyl-lysine 261.13684 Da 789.13 s 13.15 C10H19N3O5 262.14411 3-Methylindole 131.07356 Da 846.71 s 14.11 C9H9N 132.08083 Linoleic acid 280.24101 Da 998.79 s 16.65 C18H32O2 281.24828 Spermine 202.21571 Da 57.79 s 0.96 C10H26N4 203.22299 Glycerophosphocholine 257.10280 Da 73.03 s 1.22 C8H20NO6P 258.11008 L-Glutamic acid 147.05315 Da 78.63 s 1.31 C5H9NO4 148.06043 Cytidine monophosphate 323.05245 Da 85.14 s 1.42 C9H14N3O8P 324.05973 Omeprazole 345.11695 Da 96.68 s 1.61 C17H19N3O3S 346.12422 Hypoxanthine 136.03858 Da 98.93 s 1.65 C5H4N4O 137.04579 Uridine diphosphate-N-acetylglucosamine 607.08420 Da 100.61 s 1.68 C17H27N3O17P2 608.09148 Uracil 112.02700 Da 106.38 s 1.77 C4H4N2O2 113.03427 Uridine 5_-monophosphate 324.03634 Da 133.45 s 2.22 C9H13N2O9P 325.04361 Glutathione 307.08446 Da 146.40 s 2.44 C10H17N3O6S 308.09174 L-Leucine 131.09482 Da 212.85 s 3.55 C6H13NO2 132.1021 Adenosine 267.09682 Da 303.98 s 5.07 C10H13N5O4 268.1041 L-Phenylalanine 165.07894 Da 342.84 s 5.71 C9H11NO2 166.08621 5_-Methylthioadenosine 297.08999 Da 356.54 s 5.94 C11H15N5O3S 298.09727 L-Tryptophan 204.08969 Da 364.92 s 6.08 C11H12N2O2 205.09697 2-Ethyl-2-hydroxybutyric acid 132.07829 Da 379.84 s 6.33 C6H12O3 133.08557 Isovalerylcarnitine 245.16249 Da 417.22 s 6.95 C12H23NO4 246.16976 Sphingosine 299.28329 Da 752.59 s 12.54 C18H37NO2 282.27997 PC_18:1_9Z_18:1_9Z_ 785.59168 Da 1247.08 s 20.78 C44H84NO8P 786.59895 PC_16:0_16:0_ 733.56509 Da 1267.52 s 21.13 C40H80NO8P 734.57237 METABOLITES_END #END