#METABOLOMICS WORKBENCH sdhungana_20140601_9004811_mwtab.txt DATATRACK_ID:101 STUDY_ID:ST000055 ANALYSIS_ID:AN000093 PROJECT_ID:PR000052 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Metabonomic profiling of kinase inhibitor response in leukemia PR:PROJECT_TYPE Effects of kinase inhibitors in acute myeloid leukemia to understand biohemical PR:PROJECT_TYPE of disease progression and drug resistance PR:PROJECT_SUMMARY The goal of this pilot project is to develop the metabonomic parameters PR:PROJECT_SUMMARY to comprehensively profile and analyze large numbers of metabolites and their PR:PROJECT_SUMMARY responses in cell and animal models of leukemia. Our long-term objective of PR:PROJECT_SUMMARY studies is to investigate the effects of select kinase inhibitors in acute PR:PROJECT_SUMMARY leukemia (AML) in an effort to better understand biochemical mechanisms of PR:PROJECT_SUMMARY progression and drug resistance. Our focus will be on FLT3-ITD kinase positive PR:PROJECT_SUMMARY because this is one of the most aggressive and drug-resistant types of AML. PR:PROJECT_SUMMARY because of the central role of the MEK/Erk kinase pathway in regulating cell PR:PROJECT_SUMMARY and survival in these cells, inhibitors of MEK and other kinases will be tested PR:PROJECT_SUMMARY their effects on human and mouse metabolic profiles. We have developed a unique PR:PROJECT_SUMMARY of drug-resistant AML and the metabolic response of these cells to kinase PR:PROJECT_SUMMARY will also be evaluated. Cells will be treated with kinase inhibitors for varied PR:PROJECT_SUMMARY the cells isolated and the cellular metabolites identified and quantified by PR:PROJECT_SUMMARY spectrometry and NMR-based analytical methods. Bioinformatics and statistics PR:PROJECT_SUMMARY be performed and a comprehensive metabonomic analysis of metabolite profiles PR:PROJECT_SUMMARY be accomplished. Metabonomic responses will be related to specific changes in PR:PROJECT_SUMMARY signaling networks. A second major objective will be to perform metabonomic PR:PROJECT_SUMMARY in response to targeted kinase inhibitors in a mouse model of FLT3-ITD AML. We PR:PROJECT_SUMMARY measure metabonomic profiles in mouse biofluids before and after exposure to PR:PROJECT_SUMMARY inhibitors. The specific effects of MEK or other kinase inhibitors on PR:PROJECT_SUMMARY metabolites will be quantified and analyzed by bioinformatics. We anticipate PR:PROJECT_SUMMARY these studies will lead to the identification of unique sites of intersection PR:PROJECT_SUMMARY cell signaling and cell metabolism and provide the foundation for future PR:PROJECT_SUMMARY research projects. PR:INSTITUTE University of North Carolina at Chapel Hill PR:DEPARTMENT Department of Pharmacology PR:LABORATORY Graves Laboratory PR:LAST_NAME Graves PR:FIRST_NAME Lee PR:ADDRESS Pharmacology, CB7365, 411 GMB Mason Farm Road, Chapel Hill, NC 27599 PR:EMAIL LMG@med.unc.edu PR:PHONE 919-966-0915 #STUDY ST:STUDY_TITLE Effect of kinase inhibitors on FLT3-ITD AML cell metabolomes ST:STUDY_TYPE Metabolomics analysis on the effect of kinase inhibitors on FLT3-ITD AML cell ST:STUDY_TYPE to identify changes in cell signaling networks ST:STUDY_SUMMARY FLT3-ITD AML cells (MR2) obtained from mice were treated with two MEK kinase ST:STUDY_SUMMARY (GSK and AZD) at 10 uM versus media only control. Conditioned media aliquots ST:STUDY_SUMMARY cellular fractions comprised of two aliquots (tecnical replicates) for LC-MS ST:STUDY_SUMMARY and one for Western blot analyses were collected at 0, 4, 24, and 48 hours. The ST:STUDY_SUMMARY was repeated three times. HPLC-MS data were acquired for 24 samples from one ST:STUDY_SUMMARY experiment. ST:INSTITUTE RTI International ST:DEPARTMENT Systems and Translational Sciences (STS) ST:LABORATORY RTI Regional Comprehensive Metabolomics Resource Core (RTI RCMRC) ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 3040 East Cornwallis Road, Research Triangle Park, NC 27709, USA ST:EMAIL ssumner@rti.org ST:PHONE 919-541-7479 ST:NUM_GROUPS 12 ST:TOTAL_SUBJECTS 24 #SUBJECT SU:SUBJECT_TYPE Cell SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN FLT3-ITD AML (MR2) SU:CELL_BIOSOURCE_OR_SUPPLIER Dr. Tim Pardee (Wake Forest Univ.) SU:CELL_PRIMARY_IMMORTALIZED Primary SU:SPECIES_GROUP Mammal #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - MR2_AZD_205 Treatment:AZD inhibitor | Collection Time (h):0 SUBJECT_SAMPLE_FACTORS - MR2_AZD_206 Treatment:AZD inhibitor | Collection Time (h):0 SUBJECT_SAMPLE_FACTORS - MR2_GSK_213 Treatment:GSK inhibitor | Collection Time (h):0 SUBJECT_SAMPLE_FACTORS - MR2_GSK_214 Treatment:GSK inhibitor | Collection Time (h):0 SUBJECT_SAMPLE_FACTORS - MR2_CTL_197 Treatment:Media alone | Collection Time (h):0 SUBJECT_SAMPLE_FACTORS - MR2_CTL_198 Treatment:Media alone | Collection Time (h):0 SUBJECT_SAMPLE_FACTORS - MR2_AZD_229 Treatment:AZD inhibitor | Collection Time (h):4 SUBJECT_SAMPLE_FACTORS - MR2_AZD_230 Treatment:AZD inhibitor | Collection Time (h):4 SUBJECT_SAMPLE_FACTORS - MR2_GSK_237 Treatment:GSK inhibitor | Collection Time (h):4 SUBJECT_SAMPLE_FACTORS - MR2_GSK_238 Treatment:GSK inhibitor | Collection Time (h):4 SUBJECT_SAMPLE_FACTORS - MR2_CTL_221 Treatment:Media alone | Collection Time (h):4 SUBJECT_SAMPLE_FACTORS - MR2_CTL_222 Treatment:Media alone | Collection Time (h):4 SUBJECT_SAMPLE_FACTORS - MR2_AZD_253 Treatment:AZD inhibitor | Collection Time (h):24 SUBJECT_SAMPLE_FACTORS - MR2_AZD_254 Treatment:AZD inhibitor | Collection Time (h):24 SUBJECT_SAMPLE_FACTORS - MR2_GSK_262 Treatment:GSK inhibitor | Collection Time (h):24 SUBJECT_SAMPLE_FACTORS - MR2_GSK_263 Treatment:GSK inhibitor | Collection Time (h):24 SUBJECT_SAMPLE_FACTORS - MR2_CTL_245 Treatment:Media alone | Collection Time (h):24 SUBJECT_SAMPLE_FACTORS - MR2_CTL_246 Treatment:Media alone | Collection Time (h):24 SUBJECT_SAMPLE_FACTORS - MR2_AZD_279 Treatment:AZD inhibitor | Collection Time (h):48 SUBJECT_SAMPLE_FACTORS - MR2_AZD_280 Treatment:AZD inhibitor | Collection Time (h):48 SUBJECT_SAMPLE_FACTORS - MR2_GSK_288 Treatment:GSK inhibitor | Collection Time (h):48 SUBJECT_SAMPLE_FACTORS - MR2_GSK_289 Treatment:GSK inhibitor | Collection Time (h):48 SUBJECT_SAMPLE_FACTORS - MR2_CTL_270 Treatment:Media alone | Collection Time (h):48 SUBJECT_SAMPLE_FACTORS - MR2_CTL_271 Treatment:Media alone | Collection Time (h):48 #COLLECTION CO:COLLECTION_SUMMARY Conditioned Medias:Cellular Fraction_LC-MS:Cellular Fraction_WB CO:COLLECTION_PROTOCOL_COMMENTS Processed simultaneously/time point:Processed simultaneously/time CO:COLLECTION_PROTOCOL_COMMENTS simultaneously/time point CO:SAMPLE_TYPE conditioned treatment media CO:COLLECTION_METHOD Total contents pipette-transferred to tubes & centrifuged at 1800 rpm for 5 CO:COLLECTION_METHOD Medias transferred into aliquot tubes.:Cellular fractions pelleted & CO:COLLECTION_METHOD in 1X PBS. Aliqotted into collection vials based on cell counts. Re-centrifuged CO:COLLECTION_METHOD 8,000 rpm for 3 min at 4 °C. PBS was aspirated off pellets for CO:COLLECTION_METHOD fractions pelleted & resuspended in 1X PBS. Aliqotted into collection vial CO:COLLECTION_METHOD on cell counts. Re-centrifuged at 8,000 rpm for 3 min at 4 °C. PBS was CO:COLLECTION_METHOD off pellets for storage. CO:COLLECTION_LOCATION RTI RCMRC Tissue culture suite:RTI RCMRC Wet lab:RTI RCMRC Wet lab CO:COLLECTION_FREQUENCY 0h, 4h, 24h and 48h:0h, 4h, 24h and 48h:0h, 4h, 24h and 48h CO:COLLECTION_DURATION 10 minutes:10 minutes:5 minutes CO:COLLECTION_TIME 0h, 4h, 24h and 48h:0h, 4h, 24h and 48h:0h, 4h, 24h and 48h CO:VOLUMEORAMOUNT_COLLECTED 1 mL x 5 aliquots:2x10^7 cells:2x10^6 cells CO:STORAGE_CONDITIONS -80C:-80C:-80C CO:COLLECTION_VIALS 15 mL conical tubes:15 mL conical tubes:15 mL conical tubes CO:STORAGE_VIALS 1.5 mL Eppendorf Protein Lo-Bind tubes:1.5 mL Eppendorf Protein Lo-Bind CO:STORAGE_VIALS mL Eppendorf Protein Lo-Bind tubes CO:COLLECTION_TUBE_TEMP chilled in ice:chilled in ice:chilled in ice #TREATMENT TR:TREATMENT_SUMMARY MEK kinase inhibitors TR:TREATMENT_COMPOUND GSK | AZD TR:TREATMENT_ROUTE added to media TR:TREATMENT_DOSE 10nM | 10nM TR:TREATMENT_VEHICLE dilutions made in 1X sterile PBS (orginally dissolved in DMSO) TR:CELL_STORAGE liquid nitrogen, live maintenance in 5% CO2, humidified incubator TR:CELL_GROWTH_CONTAINER T-75 culture flask TR:CELL_GROWTH_CONFIG suspension TR:CELL_GROWTH_RATE double ~every 20 hours TR:CELL_MEDIA 50:50 DMEM:IMDM + 10% FBS, 1% P/S TR:CELL_HARVESTING Transfer 1 mL to new flask containing 10 mL of fresh culture media TR:CELL_PCT_CONFLUENCE maintained at ~ 2 x10^6/mL #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were extracted using 90:10 methanol:chloroform. For reversed phase, SP:SAMPLEPREP_SUMMARY were dried down and reconstituted in 95:5 water:methanol. For HILIC, samples SP:SAMPLEPREP_SUMMARY directly transferred to autosampler vials. SP:SAMPLEPREP_PROTOCOL_FILENAME RTI_GRAVES_Metabolomics_Procedure.docx SP:EXTRACTION_METHOD 90:10 methanol:chloroform SP:EXTRACT_STORAGE -80 C SP:SAMPLE_RESUSPENSION 95:5 Water:Methanol SP:SAMPLE_SPIKING L-Tryptophan-d5 SP:CELL_TYPE FLT3-ITD AML (myeloid) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity HSS T3 (100 x 2.1mm, 1.8um) CH:COLUMN_PRESSURE 6000-10000 CH:COLUMN_TEMPERATURE 50 C CH:FLOW_GRADIENT Time(min) Flow Rate %A %B Curve 1. Initial 0.400 100.0 0.0 2. CH:FLOW_GRADIENT 0.400 100.0 0.0 6 3. 16.00 0.400 0.0 100.0 6 4. 20.00 0.400 0.0 100.0 CH:FLOW_GRADIENT 5. 22.00 0.400 100.0 0.0 6 CH:FLOW_RATE 0.4 mL/min CH:INJECTION_TEMPERATURE 8 C CH:INTERNAL_STANDARD L-Tryptophan-d5 CH:SOLVENT_A Water w/ 0.1% formic acid CH:SOLVENT_B Methanol w/ 0.1% formic acid CH:ANALYTICAL_TIME 22 min CH:WEAK_WASH_SOLVENT_NAME 5%MeOH CH:WEAK_WASH_VOLUME 1000 uL CH:STRONG_WASH_SOLVENT_NAME 80%MeOH CH:STRONG_WASH_VOLUME 1000 uL CH:TARGET_SAMPLE_TEMPERATURE 8 C CH:SAMPLE_LOOP_SIZE 10 uL CH:SAMPLE_SYRINGE_SIZE 100 uL CH:RANDOMIZATION_ORDER Yes #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME RTI AN:SOFTWARE_VERSION MassLynx 4.1 AN:OPERATOR_NAME Suraj Dhungana, Darya Cheng AN:DETECTOR_TYPE TOF #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_TEMPERATURE 110 C MS:CAPILLARY_VOLTAGE 3.2 kV MS:COLLISION_ENERGY 4 MS:HELIUM_FLOW 180 MS:IONIZATION ES+ MS:MASS_ACCURACY 10 ppm MS:SOURCE_TEMPERATURE 110 C MS:DATAFORMAT Continuum MS:DESOLVATION_GAS_FLOW 400 L/Hr MS:DESOLVATION_TEMPERATURE 400 C MS:RESOLUTION_SETTING 18000 MS:SCAN_RANGE_MOVERZ 50-1000 m/s MS:SCANNING_CYCLE 1 s MS:TUBE_LENS_VOLTAGE 75 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Normalized abundance MS_METABOLITE_DATA_START Samples MR2_GSK_213 MR2_GSK_214 MR2_GSK_237 MR2_GSK_238 MR2_GSK_262 MR2_GSK_263 MR2_GSK_288 MR2_GSK_289 MR2_AZD_205 MR2_AZD_206 MR2_AZD_229 MR2_AZD_230 MR2_AZD_253 MR2_AZD_279 MR2_AZD_280 MR2_CTL_197 MR2_CTL_198 MR2_CTL_221 MR2_CTL_222 MR2_CTL_245 MR2_CTL_246 MR2_CTL_270 MR2_CTL_271 Factors Treatment:GSK inhibitor | Collection Time (h):0 Treatment:GSK inhibitor | Collection Time (h):0 Treatment:GSK inhibitor | Collection Time (h):4 Treatment:GSK inhibitor | Collection Time (h):4 Treatment:GSK inhibitor | Collection Time (h):24 Treatment:GSK inhibitor | Collection Time (h):24 Treatment:GSK inhibitor | Collection Time (h):48 Treatment:GSK inhibitor | Collection Time (h):48 Treatment:AZD inhibitor | Collection Time (h):0 Treatment:AZD inhibitor | Collection Time (h):0 Treatment:AZD inhibitor | Collection Time (h):4 Treatment:AZD inhibitor | Collection Time (h):4 Treatment:AZD inhibitor | Collection Time (h):24 Treatment:AZD inhibitor | Collection Time (h):48 Treatment:AZD inhibitor | Collection Time (h):48 Treatment:Media alone | Collection Time (h):0 Treatment:Media alone | Collection Time (h):0 Treatment:Media alone | Collection Time (h):4 Treatment:Media alone | Collection Time (h):4 Treatment:Media alone | Collection Time (h):24 Treatment:Media alone | Collection Time (h):24 Treatment:Media alone | Collection Time (h):48 Treatment:Media alone | Collection Time (h):48 0.82_130.0515m/z 0 0 48.15975846 59.14033042 49.95451384 51.96262617 23.20915164 29.11809182 2.470035526 0 36.20675901 40.26288794 50.99912914 4.50991637 8.355596441 0 0 50.04010286 43.4307967 36.13053456 69.64316393 3.459524315 3.969177548 0.82_239.1082m/z 124.6774583 124.7610413 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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 12.68189002 0 0 8.62_190.0981n 128.6179238 117.9915161 109.4915245 136.5121946 131.0988731 121.3777805 120.690345 85.67365402 134.940758 135.0813556 170.7458983 128.3057708 130.5691195 111.9963533 147.0061159 128.5424465 120.1826071 153.600106 170.5329374 108.0867945 138.4406761 79.65945609 121.5082552 8.93_163.0420m/z 63.08304981 65.22791841 63.793873 73.56052233 53.80941486 39.5333514 43.06508384 50.28881186 68.57774503 70.78584722 83.86950703 74.09739669 55.61832267 56.59670561 46.31265993 70.46253875 67.08302108 65.23788936 76.78159348 29.09291174 55.97359313 40.49673103 60.359513 9.05_145.9783m/z 1.179777409 2.026084602 3.419899151 2.254729292 0.93495267 2.177376588 0.886709799 1.089763298 5.312128934 0.943760765 2.63542296 0.042100043 0 0 1.323724597 0 0 0 2.513122207 0 0 0.170286689 1.470321717 9.05_209.9301m/z 20.3378808 21.19485556 28.74548463 27.74405076 21.95054313 21.85024477 17.18260305 19.22535012 24.42768542 16.38441247 31.47971752 32.66926196 15.55706052 20.61366631 13.71465902 26.36969359 34.40977274 18.30052545 34.23286744 10.14765944 28.85341106 11.06201953 26.2532601 9.08_355.0656m/z 23.22388188 30.44302483 25.75915144 31.49880912 55.28959065 38.59807712 45.73423043 65.51676994 38.42364265 34.35801753 30.42201552 32.74360661 15.77827387 58.76102495 70.18805797 37.77702241 33.29534416 14.2002428 23.8273158 34.60890042 38.89474877 62.36200742 68.5545607 9.17_903.9606m/z 0 2.876660938 10.91937538 17.11460936 16.45190892 4.891893341 30.05329956 0 13.58355581 24.27768097 29.57721851 0 0 12.97548844 13.59502171 3.079795024 6.121084883 19.28319411 13.158607 0 7.525629695 18.40039502 3.285683589 9.23_620.8441m/z 17.7309055 5.200674563 6.924316386 24.62658458 15.59845823 23.51060643 51.34286048 8.141032615 33.40067734 27.4617644 30.11973265 5.196410766 4.674458561 31.33713856 37.99190392 19.27975573 11.36452811 18.35203012 21.6102534 29.84439688 3.868005783 21.19544283 5.349114367 9.25_631.8335m/z 13.97905769 0 5.031679566 9.61747669 14.18640425 13.62408879 53.19565062 0 34.15341324 29.10647221 25.78020405 0 4.408886511 27.73004344 49.82802306 17.30548052 8.295394761 13.9255253 15.18300779 23.2193684 0 19.76132392 0 9.56_376.2043m/z 37.65411707 46.23516088 35.72168699 54.82434776 46.24888103 37.9283453 42.4263417 36.210708 43.96514848 32.47062059 48.21389896 46.76982437 44.84814155 30.9996897 36.53053886 46.79563933 45.86476431 42.18234903 53.57286622 24.69378991 41.7508278 32.8872663 40.1855127 9.58_269.1378m/z 60.14701346 5.962229435 6.936457354 7.04852611 75.57734694 0 20.74731439 0 80.5704133 11.15242768 90.82066285 0 71.5163931 20.86279718 0 81.21028881 2.86904891 6.749308758 0 52.70722771 0 60.73095457 6.032238965 9.58_725.3751m/z 139.8581529 192.1874196 210.9815053 229.2178097 329.426296 288.7593629 286.638258 199.8747364 329.0473219 286.9966052 179.8147762 148.9019776 175.6324667 169.9999811 215.1129716 229.6526299 194.1175521 301.7510191 222.3570363 193.3558526 241.1419958 159.8687334 169.4541176 9.59_544.7838m/z 36.40896994 50.24948533 52.21717616 57.73540339 96.58768925 89.8918076 73.03023699 42.09439457 81.79417634 79.71914768 58.29634663 46.00453834 42.31880325 46.78372409 53.4578812 58.37131318 56.80229486 73.25301209 51.60733761 69.06623014 61.18498632 37.44580173 47.92951227 9.59_725.7133m/z 116.846736 187.6128951 193.7777192 232.2506149 320.4001647 303.931562 235.023332 172.550072 294.0667292 276.7564317 145.4190003 170.0148148 192.959714 142.9067568 170.2244724 262.8576296 153.2868851 284.8603658 214.1697708 204.1349544 235.9655759 151.101866 195.9028093 9.63_726.0437m/z 79.66467224 100.2279253 108.5914046 140.7676439 186.5930483 178.5339043 129.6401217 118.1937677 195.1054438 180.3923738 67.08094288 91.63461227 83.81724514 110.0447905 109.6044055 130.4112913 120.3388885 145.5724683 105.9010297 122.1798939 137.7503988 98.35961175 93.72787272 9.65_744.3849m/z 0 10.38983919 15.49290997 9.145509647 52.01237047 44.67413234 33.16385004 35.76585937 28.43158362 16.66153172 11.00167619 11.26996348 14.98548285 16.18035143 36.22128246 4.628901225 11.22496788 32.13467606 25.965069 32.31937921 37.52134905 35.78177827 31.14818613 9.71_160.0440m/z 38.15977758 48.84816203 31.47645709 43.68994326 88.68981418 69.51399827 52.03923664 61.18384603 55.41502298 60.91260335 48.61910716 43.81916197 55.30525602 50.19449516 58.59894796 53.14948104 44.09168806 31.64503886 43.12959868 56.95541635 77.84523849 42.66176749 69.26831787 9.71_176.0716m/z 47.7963542 57.23704775 47.91473626 50.85234791 116.5231913 68.88191982 57.35154686 73.82386938 72.76162671 62.83348248 45.89894698 51.71921959 63.36134859 53.29213222 70.91163467 70.32867231 55.02741015 37.6241733 44.30996135 63.58888485 98.2916802 51.32545795 69.17871513 9.85_331.2111m/z 0 2.374248761 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4.273084629 0 69.5531403 0 0 9.89_231.1010m/z 8.726773372 20.74515479 10.62847972 26.89553008 7.947122159 21.49874787 8.042461206 17.79300786 11.47091288 14.90227083 15.70327731 10.21163807 12.23616279 3.213156153 19.08791718 20.33362406 22.28612451 0 26.50063921 9.351200416 14.01725546 5.669704051 32.08964942 9.90_277.1434m/z 2.326680285 0 0 0 4.931382696 0 0 5.154520473 2.66077796 0 0 0 1.325698786 1.285711592 0 0 0 0 0 0 97.48743264 2.092695951 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START Compound Quant m/z Ret. Index Other ID Spectra Kegg PubChem MR2_AZDpool1 MR2_AZDpool2 MR2_AZDpool3 MR2_GSKpool1 MR2_GSKpool2 MR2_GSKpool3 MR2_CTLpool1 MR2_CTLpool2 MR2_CTLpool3 0.82_130.0515m/z 130.0514877 0.82 19.71230156 14.8392565 10.80235126 26.24050743 29.19115584 20.43829192 30.68780954 24.14655523 20.79261919 0.82_239.1082m/z 239.1082489 0.82 112.0818306 127.1755134 94.42464225 98.42416277 116.7523403 98.44266442 106.2833045 115.5761138 100.656809 0.82_276.1209m/z 276.1209153 0.82 6.857557368 2.772255105 4.110310613 11.27525328 11.37955002 13.94488026 13.40341431 7.073562438 8.173577408 0.82_98.9855m/z 98.98549328 0.82 56.11717245 38.11298309 28.39046704 50.15543158 45.46363903 31.22686183 44.81422608 41.18459077 30.25242708 0.84_157.0126m/z 157.0125549 0.84 16.46216581 14.01048839 12.98837203 21.19128222 10.35213547 16.00682062 18.77723137 21.72366456 14.88283585 0.91_118.0880m/z 118.087997 0.91 152.9645247 149.770292 120.3493642 120.4591784 127.3026568 122.3006921 133.5348264 125.8070938 109.4821302 0.91_72.0817m/z 72.08174755 0.91 70.46526581 69.79799812 55.45100145 53.43112302 54.87106564 63.92821026 63.00515338 61.56154418 55.89344935 0.94_608.0916m/z 608.0915899 0.94 49.06830704 45.64091496 30.29714467 46.97658157 49.16854518 45.56883631 66.26601701 62.95314321 37.16967344 1.01_244.0949m/z 244.0949486 1.01 34.54398765 43.88190018 47.88486834 44.23318295 52.97586725 36.48363506 42.3918381 55.75370232 53.15709218 1.03_112.0519m/z 112.0519166 1.03 146.3125815 159.1155043 149.3428869 143.8439333 162.8240904 135.4404044 156.9354491 169.896655 140.9433014 1.03_487.1798m/z 487.1797576 1.03 28.60460957 31.91879902 44.84051493 21.92669039 46.62354277 45.18741691 46.44436268 50.51560408 48.14898052 1.13_152.0027n 174.9924551 1.13 342.2764882 349.4603263 293.3936806 310.1852742 320.9849688 312.5738318 303.0901505 335.2337367 291.2922775 1.15_127.9796n 128.9873753 1.15 194.1073973 186.5307649 169.2910056 143.1596791 178.2225918 159.8197302 159.1528938 175.0325708 152.3459225 1.15_144.9602m/z 144.960152 1.15 47.31428516 56.42428146 42.64809728 43.78068575 43.76590007 49.6767117 41.99777431 56.21935927 37.8980054 1.15_196.0283n 219.0193774 1.15 322.4794302 332.8473939 281.6923678 271.2686293 298.0446551 290.9669001 291.5098406 317.8823952 284.6697061 1.15_201.0089m/z 201.0088585 1.15 90.99481942 94.71220705 83.81991422 70.18858866 91.20742042 75.69770896 88.01192385 91.16712475 80.25800041 1.15_205.0046n 188.9858807 1.15 108.2589433 105.8204964 95.65578573 91.91894156 100.2467824 89.40900267 89.85282014 112.7948021 87.77481686 1.16_119.0184m/z 119.0183596 1.16 19.29257671 21.42818519 15.94207222 14.74820283 16.32620109 17.46863651 16.84798179 17.66372476 18.47675659 1.16_204.1249m/z 204.1248775 1.16 66.5933523 80.8849787 61.25965065 79.74174933 96.31008232 86.82935232 67.09631648 67.41975281 73.86609391 1.18_104.0542m/z 104.0541744 1.18 45.28923247 44.01285924 32.59438655 38.74836565 33.46165163 35.7779474 36.11070474 33.01078034 37.76960416 1.18_132.0247n 150.0590928 1.18 259.4372382 264.0340356 212.413862 193.7947317 221.4119256 206.0157036 188.4691327 204.8410914 182.4467575 1.18_133.0335m/z 133.0335219 1.18 278.0861602 280.3911607 248.5219699 202.8654964 230.5947792 229.7354706 220.975503 213.3072483 224.7335101 1.18_134.1193m/z 134.1192517 1.18 232.1894421 215.0681098 186.103912 190.2010167 199.8710729 196.5865146 246.3476262 270.0976569 235.4577218 1.18_172.0037n 205.0304544 1.18 133.1536007 133.0777412 125.5793287 103.1486571 102.8394192 122.0067916 126.521181 130.2712482 117.6561661 1.27_668.1072m/z 668.1072344 1.27 0 0 4.136996614 0 0 0 6.852751001 0 3.77189058 1.28_161.0161n 179.0496523 1.28 724.6112871 743.5723774 635.1816423 686.5913284 712.6827122 719.8940217 957.0093619 863.950019 922.0323063 1.28_233.0609m/z 233.0608943 1.28 237.1086346 247.6901956 196.8112277 213.4715433 222.5209908 220.8617124 285.5400549 286.8487441 285.9279117 1.28_245.0632m/z 245.0631812 1.28 48.25272076 46.18684227 41.87119492 41.88770814 49.0764496 49.27927415 59.91566849 48.15428282 50.40270306 1.28_307.0844n 308.0922625 1.28 6310.828337 6150.702566 5333.024052 5383.958468 5957.327512 6026.363 8435.998911 7103.289549 7878.430456 1.28_362.0104m/z 362.0104269 1.28 15.53491187 19.89108725 9.909932435 10.46542718 11.14085345 12.59329615 15.19921245 21.07364871 20.84328389 1.28_615.1784m/z 615.1784202 1.28 29.84052923 24.08149504 23.43940546 20.89701467 25.19194176 20.58673221 50.54048726 52.55342246 56.23189935 1.28_76.0228m/z 76.02282931 1.28 22.75665904 24.59499687 22.9574954 21.67889146 21.83058612 23.81921229 31.92515603 29.68153232 37.01538868 1.32_348.0719m/z 348.0718521 1.32 126.8845723 134.1276457 124.7767925 181.746168 231.1012304 221.1175218 342.2465694 360.693867 363.334217 1.40_169.0393m/z 169.0392906 1.4 42.5789263 48.11005827 39.26380052 37.31883337 38.6041553 37.2442464 41.78774269 39.38888294 40.46442976 1.45_123.0565m/z 123.0565335 1.45 1047.231033 1090.137056 956.1399249 983.0301525 1001.390179 1031.067546 1032.798552 1072.484579 1047.364339 1.47_152.0578m/z 152.0578002 1.47 9.956117992 7.042622837 4.151988571 26.20003485 19.53761576 25.16403253 46.31401342 34.38914243 40.28280079 1.53_136.0393n 137.0471439 1.53 3594.528845 4474.494032 3811.150394 3508.097838 4409.690952 4503.046794 3051.039676 3482.386799 3358.017498 1.61_130.0515m/z 130.0515332 1.61 264.7330876 268.8508121 210.9694435 224.8609475 222.7643535 219.840617 310.5566319 282.6748751 280.0154229 1.63_191.0129n 229.976562 1.63 119.7142669 118.168611 87.79794189 104.4620131 120.5473131 106.0578975 118.4424396 126.7416798 123.7457329 1.63_84.0457m/z 84.0457393 1.63 41.35534418 42.54576116 40.66093449 39.31109003 38.26186621 40.22865264 53.46702228 51.54102558 48.80501967 1.73_290.1367m/z 290.136741 1.73 94.08525771 91.65225759 88.01241823 116.7320746 138.7490794 120.4870623 88.100659 82.90046598 73.57355373 1.83_156.9826m/z 156.9826238 1.83 19.30012162 19.45015991 18.91373286 19.25219988 22.35901236 21.47887116 24.46653535 29.37903674 23.48837942 1.87_152.0343n 153.0421474 1.87 851.978776 825.0721217 695.7099157 683.8211758 716.5514316 682.4068824 698.4773259 642.226322 660.306049 1.88_123.0455m/z 123.0454513 1.88 257.1817004 250.9468805 208.2433061 197.3768583 210.7406163 205.6781979 200.3605873 204.3633966 196.5258633 1.88_135.0696n 136.0774227 1.88 905.1200614 887.8755725 739.9891408 677.5315177 744.3454746 722.0699047 697.9530854 677.6868919 668.0332961 1.88_164.0483n 165.0558147 1.88 1872.330834 1838.365434 1580.220832 1380.651656 1526.905086 1467.730555 1458.12504 1411.597891 1371.187364 1.88_91.0554m/z 91.05539544 1.88 18.44608468 21.91449002 16.91263344 14.49525094 11.95647465 14.06068427 14.34436805 18.32778812 9.008512052 1.99_132.1036m/z 132.1036041 1.99 317.0371206 271.4272275 232.6517938 222.2173181 250.3425761 246.4484384 280.6147007 259.8055662 220.0761191 10.42_229.1689n 252.1587004 10.42 55.88060724 59.6081134 60.17820378 62.5884219 79.3473101 78.70169928 87.62526887 117.1162379 119.3624562 10.42_892.7959m/z 892.7958837 10.42 4.905213447 0 0 0 1.913701755 4.974707286 3.989345937 14.62353086 0 10.50_331.2061m/z 331.2060626 10.5 18.38441541 17.57475368 8.217877684 34.65464094 38.1646556 17.12935944 9.252732692 15.96858399 11.43175413 10.55_124.9799n 125.9877596 10.55 266.7854905 343.304417 289.420761 294.2572789 334.4291898 317.6194636 282.5365734 323.2869424 306.5181136 10.60_728.6919m/z 728.6919058 10.6 0 0 4.552113764 10.86302964 8.8858227 14.40305049 0 3.40807377 0 10.62_930.4857m/z 930.4857285 10.62 7.847991943 21.28903452 23.0445833 14.66686649 31.79518672 46.59297994 20.23492858 50.56709788 52.33175678 10.62_930.8265m/z 930.8265014 10.62 23.26474675 40.51853637 32.26244531 38.8232203 40.11257051 58.55200886 29.03414066 55.87943507 40.03305384 10.69_237.1466m/z 237.1466032 10.69 12.83138869 23.42785176 26.60383537 15.91105462 25.08968133 23.71221822 17.23817281 20.02261423 23.33827826 10.74_163.0393m/z 163.0392589 10.74 86.59533708 103.6938088 74.17998385 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