#METABOLOMICS WORKBENCH agoldman_20240429_061721 DATATRACK_ID:4798 STUDY_ID:ST003182 ANALYSIS_ID:AN005226 PROJECT_ID:PR001981 VERSION 1 CREATED_ON April 30, 2024, 11:39 am #PROJECT PR:PROJECT_TITLE METTL3-mediated chromatin contacts promote stress granule phase separation PR:PROJECT_TITLE through metabolic reprogramming during senescence PR:PROJECT_SUMMARY METTL3 is the catalytic subunit of the methyltransferase complex, which mediates PR:PROJECT_SUMMARY m6A modification to regulate gene expression. In addition, METTL3 regulates PR:PROJECT_SUMMARY transcription in an enzymatic activity-independent manner by driving changes in PR:PROJECT_SUMMARY high-order chromatin structure. However, how these functions of MTC are PR:PROJECT_SUMMARY coordinated remains unknown. Here we show that the methyltransferase complex PR:PROJECT_SUMMARY coordinates its enzymatic activity-dependent and independent functions to PR:PROJECT_SUMMARY regulate cellular senescence, a state of stable cell growth arrest. PR:PROJECT_SUMMARY Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression PR:PROJECT_SUMMARY through the three-dimensional chromatin organization during senescence. Elevated PR:PROJECT_SUMMARY Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, PR:PROJECT_SUMMARY manifesting as stress granule phase separation, by driving metabolic PR:PROJECT_SUMMARY reprogramming. This correlates with an impairment of translation of cell-cycle PR:PROJECT_SUMMARY related mRNAs harboring polymethylated m6A sites. In summary, our results report PR:PROJECT_SUMMARY a coordination of m6A-dependent and -independent function of the PR:PROJECT_SUMMARY methyltransferase complex in regulating senescence through phase separation PR:PROJECT_SUMMARY driven by metabolic reprogramming. PR:INSTITUTE University of Texas MD Anderson Cancer Center PR:LAST_NAME Zhang PR:FIRST_NAME Rugang PR:ADDRESS 3SCR3.4121, 1901 East RD, Houston, TX, 77054 PR:EMAIL rzhang11@mdanderson.org PR:PHONE 832-748-6422 #STUDY ST:STUDY_TITLE METTL3-mediated chromatin contacts promote stress granule phase separation ST:STUDY_TITLE through metabolic reprogramming during senescence ST:STUDY_SUMMARY METTL3 is the catalytic subunit of the methyltransferase complex, which mediates ST:STUDY_SUMMARY m6A modification to regulate gene expression. In addition, METTL3 regulates ST:STUDY_SUMMARY transcription in an enzymatic activity-independent manner by driving changes in ST:STUDY_SUMMARY high-order chromatin structure. However, how these functions of MTC are ST:STUDY_SUMMARY coordinated remains unknown. Here we show that the methyltransferase complex ST:STUDY_SUMMARY coordinates its enzymatic activity-dependent and independent functions to ST:STUDY_SUMMARY regulate cellular senescence, a state of stable cell growth arrest. ST:STUDY_SUMMARY Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression ST:STUDY_SUMMARY through the three-dimensional chromatin organization during senescence. Elevated ST:STUDY_SUMMARY Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, ST:STUDY_SUMMARY manifesting as stress granule phase separation, by driving metabolic ST:STUDY_SUMMARY reprogramming. This correlates with an impairment of translation of cell-cycle ST:STUDY_SUMMARY related mRNAs harboring polymethylated m6A sites. In summary, our results report ST:STUDY_SUMMARY a coordination of m6A-dependent and -independent function of the ST:STUDY_SUMMARY methyltransferase complex in regulating senescence through phase separation ST:STUDY_SUMMARY driven by metabolic reprogramming. ST:INSTITUTE University of Texas MD Anderson Cancer Center ST:LAST_NAME Zhang ST:FIRST_NAME Rugang ST:ADDRESS 3SCR3.4121, 1901 East RD, Houston, TX, 77054 ST:EMAIL rzhang11@mdanderson.org ST:PHONE 832-748-6422 #SUBJECT SU:SUBJECT_TYPE Cultured cells 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 - Pro_1 Treatment:Pro | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Pro_1-S4.raw SUBJECT_SAMPLE_FACTORS - Pro_2 Treatment:Pro | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Pro_2-S5.raw SUBJECT_SAMPLE_FACTORS - Pro_3 Treatment:Pro | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Pro_3-S6.raw SUBJECT_SAMPLE_FACTORS - Sen_shctrl_1 Treatment:Sen_shctrl | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shctrl_1-S10.raw SUBJECT_SAMPLE_FACTORS - Sen_shctrl_2 Treatment:Sen_shctrl | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shctrl_2-S11.raw SUBJECT_SAMPLE_FACTORS - Sen_shctrl_3 Treatment:Sen_shctrl | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shctrl_3-S12.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_1 Treatment:Sen_shHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_1-S16.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_2 Treatment:Sen_shHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_2-S17.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_3 Treatment:Sen_shHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_3-S18.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_MutHK2_1 Treatment:Sen_shHK2_MutHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_MutHK2_1-S28.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_MutHK2_2 Treatment:Sen_shHK2_MutHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_MutHK2_2-S29.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_MutHK2_3 Treatment:Sen_shHK2_MutHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_MutHK2_3-S30.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_WTHK2_1 Treatment:Sen_shHK2_WTHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_WTHK2_1-S22.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_WTHK2_2 Treatment:Sen_shHK2_WTHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_WTHK2_2-S23.raw SUBJECT_SAMPLE_FACTORS - Sen_shHK2_WTHK2_3 Treatment:Sen_shHK2_WTHK2 | Sample source:lung embryonic fibroblasts RAW_FILE_NAME(raw_data)=Zhang_m22-L299_C13_Sen_shHK2_WTHK2_3-S24.raw #COLLECTION CO:COLLECTION_SUMMARY IMR90 primary human diploid lung embryonic fibroblasts were used and cultured in CO:COLLECTION_SUMMARY DMEM medium supplemented with 10% fetal bovine serum (FBS), L-glutamine, sodium CO:COLLECTION_SUMMARY pyruvate, non-essential amino acids, sodium bicarbonate and 1% CO:COLLECTION_SUMMARY penicillin-streptomycin under low oxygen tension (2%). CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Stable isotope tracer analysis using 13C6-glucose was conducted in three TR:TREATMENT_SUMMARY biologically independent experiments for the following experimental groups: TR:TREATMENT_SUMMARY control proliferating cells (Pro), RAS-induced senescent cells via 4-OHT with TR:TREATMENT_SUMMARY control shRNA (Sen_shctrl), senescent cells with shRNA targeting HK2 TR:TREATMENT_SUMMARY (Sen_shHK2), senescent cells with shRNA targeting HK2 and rescued with wildtype TR:TREATMENT_SUMMARY Flag-HK2 (Sen_shHK2_WTHK2), and senescent cells with shRNA targeting HK2 and TR:TREATMENT_SUMMARY rescued with mutant Flag-HK2 (Sen_shHK2_MutHK2). The experiments were performed TR:TREATMENT_SUMMARY by seeding cells at a density of 3 × 10^5 cells per 6 cm dish and incubating TR:TREATMENT_SUMMARY them with 25 mM [13C6]-glucose tracer in glucose-free medium for 30 min. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Following incubation, the cells were washed with chilled PBS and incubated with SP:SAMPLEPREP_SUMMARY 500 µl of extraction solution (80:20 v/v methanol/water) at 4 °C for 5 SP:SAMPLEPREP_SUMMARY minutes. Next, cells were scraped with a polypropylene cell scraper and the SP:SAMPLEPREP_SUMMARY extraction solution from each sample was collected, vortexed, and incubated on SP:SAMPLEPREP_SUMMARY dry ice for at least 30 min. Then, each sample was centrifuged at maximum speed SP:SAMPLEPREP_SUMMARY at 4 °C for 10 minutes, and the resulting supernatant was used for metabolic SP:SAMPLEPREP_SUMMARY measurements. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Hydrophilic interaction liquid chromatography (HILIC) was performed at 0.2 CH:CHROMATOGRAPHY_SUMMARY ml/min on a ZIC-pHILIC column (2.1 mm × 150 mm, 5 µm particle size, EMD CH:CHROMATOGRAPHY_SUMMARY Millipore) at 45 °C. Solvent A was 20 mM ammonium carbonate, 0.1% ammonium CH:CHROMATOGRAPHY_SUMMARY hydroxide, pH 9.2, and solvent B was acetonitrile. The gradient was 85% B for 2 CH:CHROMATOGRAPHY_SUMMARY min, 85% B to 20% B over 15 min, 20% B to 85% B over 0.1 min, and 85% B for 8.9 CH:CHROMATOGRAPHY_SUMMARY min. The autosampler was held at 4 °C. For each analysis, 4 µl of sample was CH:CHROMATOGRAPHY_SUMMARY injected. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish Horizon UHPLC CH:COLUMN_NAME Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A 100% water; 20 mM ammonium carbonate; 5 µM medronic acid; 0.1% ammonium CH:SOLVENT_A hydroxide CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 85% B for 2 min, 85% B to 20% B over 15 min, 20% B to 85% B over 0.1 min, and CH:FLOW_GRADIENT 85% B for 8.9 min CH:FLOW_RATE 0.2 ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS The following parameters were used for the MS analysis: sheath gas flow rate, MS:MS_COMMENTS 40; auxiliary gas flow rate, 10; sweep gas flow rate, 2; auxiliary gas heater MS:MS_COMMENTS temperature, 350 °C; spray voltage, 3.5 kV for positive mode and 3.2 kV for MS:MS_COMMENTS negative mode; capillary temperature, 325 °C; and funnel RF level, 40. All MS:MS_COMMENTS samples were analyzed by full MS with polarity switching. Technical injections MS:MS_COMMENTS of an unlabeled sample pool were analyzed throughout the sample sequence. The MS:MS_COMMENTS unlabeled sample pool was also analyzed by data-dependent MS/MS with separate MS:MS_COMMENTS runs for positive and negative ion modes. Full MS scans were acquired at 120,000 MS:MS_COMMENTS resolution with a scan range of 65-975 m/z. Data-dependent MS/MS scans were MS:MS_COMMENTS acquired for the top 10 highest intensity ions at 15,000 resolution with an MS:MS_COMMENTS isolation width of 1.0 m/z and stepped normalized collision energy of 20-40-60. MS:MS_COMMENTS Annotation and quantitation of metabolites and carbon isotopologues with natural MS:MS_COMMENTS isotope abundance correction were performed using Compound Discoverer 3.3 MS:MS_COMMENTS software (Thermo Scientific). Metabolite measurements were normalized based on MS:MS_COMMENTS the protein concentration in the protein pellets. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples Pro_1 Pro_2 Pro_3 Sen_shctrl_1 Sen_shctrl_2 Sen_shctrl_3 Sen_shHK2_1 Sen_shHK2_2 Sen_shHK2_3 Sen_shHK2_WTHK2_1 Sen_shHK2_WTHK2_2 Sen_shHK2_WTHK2_3 Sen_shHK2_MutHK2_1 Sen_shHK2_MutHK2_2 Sen_shHK2_MutHK2_3 Factors Treatment:Pro | Sample source:lung embryonic fibroblasts Treatment:Pro | Sample source:lung embryonic fibroblasts Treatment:Pro | Sample source:lung embryonic fibroblasts Treatment:Sen_shctrl | Sample source:lung embryonic fibroblasts Treatment:Sen_shctrl | Sample source:lung embryonic fibroblasts Treatment:Sen_shctrl | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2 | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2 | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2 | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2_WTHK2 | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2_WTHK2 | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2_WTHK2 | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2_MutHK2 | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2_MutHK2 | Sample source:lung embryonic fibroblasts Treatment:Sen_shHK2_MutHK2 | Sample source:lung embryonic fibroblasts AMP M+0 103793851.2 88596725.41 116556852.8 22540389.44 18513530.99 15425235.93 59077216.67 44547074.96 25744605.32 20267922.52 20761915.4 14014033.4 49456270.65 54673121.49 35875769.55 AMP M+1 0 0 0 0 0 0 0 0 0 0 0 12794.0613 0 0 0 AMP M+2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AMP M+3 42309.9174 0 98583.43082 0 0 0 42264.38084 0 0 17678.58843 0 0 0 0 0 AMP M+4 152763.1229 0 0 0 0 0 22626.72751 0 0 0 0 0 0 0 0 AMP M+5 6198420.242 5148233.732 7444568.757 1543652.829 1155306.142 951633.7072 2191537.353 1461186.156 683017.6103 924726.1375 1002994.159 628972.8732 2175121.724 2178905.226 1383034.862 AMP M+6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AMP M+7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AMP M+8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AMP M+9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AMP M+10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ADP M+0 102542588.3 83493331.45 87090774.85 55225875.51 44524051.39 33836081.79 114078880.2 131281362.5 79335440.71 53063303.96 61394976.39 40859321.4 81519453.8 96164920.45 62010155.79 ADP M+1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ADP M+2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ADP M+3 296341.1094 228846.9658 167343.4405 269471.0315 186565.7538 161876.634 229659.1777 234974.8276 116452.5481 401742.6352 461994.1629 236875.8474 226722.2428 202740.2479 143373.7973 ADP M+4 552504.8202 341456.982 464285.3915 128928.8035 176914.0537 123738.4141 124920.9173 235587.9403 208654.6353 247306.3452 162029.8277 184143.7103 250639.3208 266139.3152 183140.1178 ADP M+5 5977200.099 4713330.682 5366573.703 4696820.568 3317551.688 2348668.321 4293973.162 5134868.917 3052694.995 3265384.341 3522209.04 2522860.791 3358210.374 3712202.625 2458990.534 ADP M+6 0 0 0 1093.045937 3524.331493 0 0 0 0 0 0 0 0 0 0 ADP M+7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ADP M+8 0 0 0 0 0 0 71383.17642 0 0 0 0 0 0 0 0 ADP M+9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ADP M+10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Ribose-5-phosphate M+0 809875.9885 705567.0434 682873.6836 1321420.87 889312.5105 1523557.778 1472218.231 3379249.72 2603064.779 3659826.47 2183726.54 1689977.668 2263751.76 3303811.162 2496803.919 Ribose-5-phosphate M+1 0 0 0 0 31102.70667 39154.68282 0 0 0 102282.6855 9636.692402 42707.25363 0 0 0 Ribose-5-phosphate M+2 2217279.233 2075156.434 2268830.064 3275502.945 2283809.446 3316039.067 2487117.059 3965922.193 3209733.742 4054153.094 3159797.481 2941056.821 3946129.797 6258526.758 4979428.469 Ribose-5-phosphate M+3 1780246.529 1604019.69 1811458.893 3582832.988 2521097.557 3753624.066 1571382.345 3256192.495 3216950.263 4414710.827 3372212.655 2683083.498 3136538.827 4785907.111 3603059.358 Ribose-5-phosphate M+4 0 0 0 375623.681 182280.4931 618994.7562 574370.2637 323191.0377 411124.9172 840052.073 498495.9853 348622.9456 456450.8578 901644.0857 822570.9392 Ribose-5-phosphate M+5 61050510.27 53449955.01 51416870.98 38743909.96 31998700.41 35173197.16 64598852.17 90789332.16 58795482.48 58573489.36 48968899.65 40028962.73 83009909.25 98701839.6 71706534.01 Fructose-1,6-bisphosphate M+0 16180904.43 10579048.9 17597032.73 19450676.97 7750397.419 12296228.35 8230390.028 16881282.39 9472580.212 10658221.38 22026218.28 13944047.48 15812148.28 19970955.93 13743637.71 Fructose-1,6-bisphosphate M+1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Fructose-1,6-bisphosphate M+2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Fructose-1,6-bisphosphate M+3 12014315.56 14373039.71 17865011.4 16663182.19 12448365.69 11842716.82 15501292.76 20178181.21 17901570.03 13097597.54 14050882.03 8985012.527 18846360.84 27111657.7 21526459.81 Fructose-1,6-bisphosphate M+4 0 92337.93396 0 173295.7935 221615.8894 232689.6261 304525.5155 0 196789.1837 245484.3887 196176.0973 126892.5082 200671.0712 8239.842235 93047.09168 Fructose-1,6-bisphosphate M+5 418783.3633 0 2126809.319 0 261795.3973 0 2472897.163 945456.2621 1050460.128 0 460311.5853 786990.167 436912.3502 0 921045.706 Fructose-1,6-bisphosphate M+6 263103116.9 231353070 231892194.5 104064384.3 86647908.42 72346705.35 238006164 278113317 193270988.7 114695102.9 116333163.5 73985574.77 216386895.9 245413031.3 170059657.2 Glucose-6-phosphate M+0 1145202.565 889954.3844 893480.2102 694697.9387 526701.0551 610087.4828 1123142.768 2033740.997 1606947.209 1321996.815 1507613.501 881386.0075 1572711.999 1636961.534 1327183.762 Glucose-6-phosphate M+1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Glucose-6-phosphate M+2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Glucose-6-phosphate M+3 0 0 0 0 0 0 29558.46935 0 146452.2086 0 0 0 0 0 0 Glucose-6-phosphate M+4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Glucose-6-phosphate M+5 253.9248969 21870.407 0 0 0 0 17087.99061 0 4868.439955 0 0 67906.97056 0 0 0 Glucose-6-phosphate M+6 7241137.099 5118061.369 5757361.194 2087051.216 1792623.969 2004861.271 10410019.35 14859016.78 10117495.71 3737467.717 5281852.859 2680914.264 8404471.32 8337469.599 7330243.734 Adenosine M+0 76724845.08 52094434.22 56973005.33 47885041.74 30499970.3 30305905.74 119440500.3 154093307 89098245.8 78704781.9 74905253.06 39192802.77 113195609.1 122183503 100572052.7 Adenosine M+1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Adenosine M+2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Adenosine M+3 0 0 55320.99747 0 0 0 330794.9897 202235.2431 77395.82892 435314.8882 191257.7852 103367.6747 171396.7942 173571.8741 167339.5474 Adenosine M+4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Adenosine M+5 3792918.43 2287895.247 3098010.724 2703396.087 1440554.938 1636820.855 3800303.519 5200910.609 2460050.513 4172115.227 3417750.105 1722776.569 4267812.177 4448293.837 3679564.671 Adenosine M+6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Adenosine M+7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Adenosine M+8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Adenosine M+9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Adenosine M+10 0 4531.273943 0 0 0 0 4899.506161 4417.320405 1499.580077 0 0 0 2525.963205 2273.19432 1556.615202 ATP M+0 59335545.47 54305123.27 48157330.52 81393050.39 61392202.06 43303267.3 173409888.3 182609281.5 124737435 53545144.17 82068706.3 45489552.39 75849355.3 85283284.22 58221327.58 ATP M+1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ATP M+2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ATP M+3 0 0 0 259819.7715 87544.94944 2378.435709 191467.7598 0 176937.5669 182250.9165 583647.9772 202382.8718 0 64654.01831 0 ATP M+4 0 0 0 0 38712.30808 0 59801.10914 0 12087.44699 0 0 0 0 0 0 ATP M+5 3185937.859 2622896.965 2822477.038 6591287.074 4264682.328 3124950.529 5581257.986 6422162.05 4318659.439 3573780.618 4875397.595 2854832.784 2950748.82 3119150.84 2270418.545 ATP M+6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ATP M+7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ATP M+8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ATP M+9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ATP M+10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Inosine M+0 252750830.3 268477336.3 273129441.5 157620959.1 120119663.7 134993422.9 248495719.5 365708193.9 325368803.6 79821414.77 86647989.86 54450299.63 281562897.4 396146279.6 328331706.8 Inosine M+1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Inosine M+2 0 0 0 0 0 203160.4577 0 0 0 0 234238.3045 0 0 0 0 Inosine M+3 496508.6759 182134.5012 371948.1166 1523141.976 1183550.521 1539200.157 850096.0822 625807.9549 1076035.896 1139153.138 586374.3339 694471.8697 602052.1287 1796227.575 1203779.314 Inosine M+4 0 0 128812.4057 0 0 0 12163.9132 0 156411.414 372020.7961 315297.1304 0 0 42536.46097 566199.4358 Inosine M+5 25088358.01 23407274.47 27000385.88 34470883.58 27000893.45 29397959.92 18777423.99 29191366.73 25381513.31 17494005.62 17822621.18 12693868.73 26600632.67 36759211.08 32539945.6 Inosine M+6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Inosine M+7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Inosine M+8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Inosine M+9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Inosine M+10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IMP M+0 31043350.52 27493621.53 28371445.51 4249488.008 3914046.031 3377491.819 23458505.71 27836383.22 17550291.28 2383759.416 2217969.458 1723486.647 17444282.17 24831591.4 17726451.67 IMP M+1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IMP M+2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IMP M+3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IMP M+4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IMP M+5 2379937.654 1796628.342 2429529.255 459699.1948 364477.318 314128.3811 1077737.016 1587553.353 1065286.227 471461.9034 295585.5103 248897.4523 1114230.272 1572894.198 1183226.684 IMP M+6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IMP M+7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IMP M+8 0 0 180308.0595 0 0 0 434887.9736 473251.8367 298706.8704 0 0 0 721451.0962 408642.8851 601278.5799 IMP M+9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IMP M+10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT AMP M+0 8.795 AMP M+1 8.795 AMP M+2 8.795 AMP M+3 8.795 AMP M+4 8.795 AMP M+5 8.795 AMP M+6 8.795 AMP M+7 8.795 AMP M+8 8.795 AMP M+9 8.795 AMP M+10 8.795 ADP M+0 9.87 ADP M+1 9.87 ADP M+2 9.87 ADP M+3 9.87 ADP M+4 9.87 ADP M+5 9.87 ADP M+6 9.87 ADP M+7 9.87 ADP M+8 9.87 ADP M+9 9.87 ADP M+10 9.87 Ribose-5-phosphate M+0 9.887 Ribose-5-phosphate M+1 9.887 Ribose-5-phosphate M+2 9.887 Ribose-5-phosphate M+3 9.887 Ribose-5-phosphate M+4 9.887 Ribose-5-phosphate M+5 9.887 Fructose-1,6-bisphosphate M+0 11.613 Fructose-1,6-bisphosphate M+1 11.613 Fructose-1,6-bisphosphate M+2 11.613 Fructose-1,6-bisphosphate M+3 11.613 Fructose-1,6-bisphosphate M+4 11.613 Fructose-1,6-bisphosphate M+5 11.613 Fructose-1,6-bisphosphate M+6 11.613 Glucose-6-phosphate M+0 10.702 Glucose-6-phosphate M+1 10.702 Glucose-6-phosphate M+2 10.702 Glucose-6-phosphate M+3 10.702 Glucose-6-phosphate M+4 10.702 Glucose-6-phosphate M+5 10.702 Glucose-6-phosphate M+6 10.702 Adenosine M+0 4.159 Adenosine M+1 4.159 Adenosine M+2 4.159 Adenosine M+3 4.159 Adenosine M+4 4.159 Adenosine M+5 4.159 Adenosine M+6 4.159 Adenosine M+7 4.159 Adenosine M+8 4.159 Adenosine M+9 4.159 Adenosine M+10 4.159 ATP M+0 10.636 ATP M+1 10.636 ATP M+2 10.636 ATP M+3 10.636 ATP M+4 10.636 ATP M+5 10.636 ATP M+6 10.636 ATP M+7 10.636 ATP M+8 10.636 ATP M+9 10.636 ATP M+10 10.636 Inosine M+0 6.377 Inosine M+1 6.377 Inosine M+2 6.377 Inosine M+3 6.377 Inosine M+4 6.377 Inosine M+5 6.377 Inosine M+6 6.377 Inosine M+7 6.377 Inosine M+8 6.377 Inosine M+9 6.377 Inosine M+10 6.377 IMP M+0 9.88 IMP M+1 9.88 IMP M+2 9.88 IMP M+3 9.88 IMP M+4 9.88 IMP M+5 9.88 IMP M+6 9.88 IMP M+7 9.88 IMP M+8 9.88 IMP M+9 9.88 IMP M+10 9.88 METABOLITES_END #END