#METABOLOMICS WORKBENCH my265_20220715_125750_mwtab.txt DATATRACK_ID:3349 STUDY_ID:ST002227 ANALYSIS_ID:AN003636 PROJECT_ID:PR001418 VERSION 1 CREATED_ON July 15, 2022, 1:36 pm #PROJECT PR:PROJECT_TITLE Dynamic partitioning of branched-chain amino acids-derived nitrogen supports PR:PROJECT_TITLE renal cancer progression PR:PROJECT_SUMMARY Metabolic reprogramming is critical for tumor initiation and progression. PR:PROJECT_SUMMARY However, the exact impact of specific metabolic changes on cancer progression is PR:PROJECT_SUMMARY poorly understood. Here, we integrate multimodal analyses of primary and PR:PROJECT_SUMMARY metastatic clonally related clear cell renal cancer cells (ccRCC) grown in PR:PROJECT_SUMMARY physiological media to identify key stage-specific metabolic vulnerabilities. We PR:PROJECT_SUMMARY show that a VHL loss-dependent reprogramming of branched-chain amino acid PR:PROJECT_SUMMARY catabolism sustains the de novo biosynthesis of aspartate and arginine enabling PR:PROJECT_SUMMARY tumor cells with the flexibility of partitioning the nitrogen of the amino acids PR:PROJECT_SUMMARY depending on their needs. Importantly, we identify the epigenetic reactivation PR:PROJECT_SUMMARY of argininosuccinate synthase (ASS1), a urea cycle enzyme suppressed in primary PR:PROJECT_SUMMARY ccRCC, as a crucial event for metastatic renal cancer cells to acquire the PR:PROJECT_SUMMARY capability to generate arginine, invade in vitro and metastasize in vivo. PR:PROJECT_SUMMARY Overall, our study uncovers a novel mechanism of metabolic flexibility occurring PR:PROJECT_SUMMARY during ccRCC progression, paving the way for the development of novel PR:PROJECT_SUMMARY stage-specific therapies. PR:INSTITUTE CECAD Research Center PR:LAST_NAME Yang PR:FIRST_NAME Ming PR:ADDRESS Joseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany PR:EMAIL ming.yang@uni-koeln.de PR:PHONE 4922147884306 #STUDY ST:STUDY_TITLE Assessing the contribution of branched-chain amino acid (BCAA)-derived nitrogen ST:STUDY_TITLE to amino acid biosynthesis in renal cell lines HK2 and 786-O. ST:STUDY_SUMMARY The objective of this experiment is to test the contribution of branched-chain ST:STUDY_SUMMARY amino acids (BCAA)-derived nitrogen to the generation of de novo amino acids in ST:STUDY_SUMMARY renal cells through transamination reactions. To test this hypothesis, we ST:STUDY_SUMMARY incubated human renal epithelial cell line HK2 and ccRCC cell lines 786-O, ST:STUDY_SUMMARY 786-M1A and 786-M2A with 15N-leucine and 15N-isoleucine in Plasmax media for ST:STUDY_SUMMARY 27h. Data were generated from 5 independent cultures. This is Part 2 of the ST:STUDY_SUMMARY study and the experimental number is MS48. ST:INSTITUTE CECAD Research Center ST:LAST_NAME Yang ST:FIRST_NAME Ming ST:ADDRESS Joseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany ST:EMAIL ming.yang@uni-koeln.de ST:PHONE 4922147884306 #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 - MS48-01 Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-01 SUBJECT_SAMPLE_FACTORS - MS48-02 Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-02 SUBJECT_SAMPLE_FACTORS - MS48-03 Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-03 SUBJECT_SAMPLE_FACTORS - MS48-04 Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-04 SUBJECT_SAMPLE_FACTORS - MS48-05 Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-05 SUBJECT_SAMPLE_FACTORS - MS48-06 Cell line:786-O | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-06 SUBJECT_SAMPLE_FACTORS - MS48-07 Cell line:786-O | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-07 SUBJECT_SAMPLE_FACTORS - MS48-08 Cell line:786-O | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-08 SUBJECT_SAMPLE_FACTORS - MS48-09 Cell line:786-O | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-09 SUBJECT_SAMPLE_FACTORS - MS48-10 Cell line:786-O | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-10 SUBJECT_SAMPLE_FACTORS - MS49-11 Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS49-11 SUBJECT_SAMPLE_FACTORS - MS48-12 Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-12 SUBJECT_SAMPLE_FACTORS - MS48-13 Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-13 SUBJECT_SAMPLE_FACTORS - MS48-14 Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-14 SUBJECT_SAMPLE_FACTORS - MS48-15 Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-15 SUBJECT_SAMPLE_FACTORS - MS48-16 Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-16 SUBJECT_SAMPLE_FACTORS - MS48-17 Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-17 SUBJECT_SAMPLE_FACTORS - MS48-18 Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-18 SUBJECT_SAMPLE_FACTORS - MS48-19 Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-19 SUBJECT_SAMPLE_FACTORS - MS48-20 Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine RAW_FILE_NAME=MS48-20 #COLLECTION CO:COLLECTION_SUMMARY 2x105 cells were plated onto 6-well plates in Plasmax media (5 replicates for CO:COLLECTION_SUMMARY each cell type). The day after, the medium was replaced with fresh one CO:COLLECTION_SUMMARY containing 15N-leucine and 15N-isoleucine for 27h. Before extraction, cells were CO:COLLECTION_SUMMARY counted using CASY cell counter (Omni Life Sciences) using a separate counting CO:COLLECTION_SUMMARY plate. After that, cells were washed at room temperature with PBS twice and then CO:COLLECTION_SUMMARY kept in a cold bath with dry ice and methanol before adding the metabolite CO:COLLECTION_SUMMARY extraction solution. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Cells were cultured in Plasmax media supplemented with 2.5% FBS in the presence TR:TREATMENT_SUMMARY of 15N-leucine and 15N-isoleucine for 27 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure SP:SAMPLEPREP_SUMMARY water, 5 µM final concentration valine-d8) was added to each well after the SP:SAMPLEPREP_SUMMARY washes in PBS following the proportion of 1ml of extraction solution per million SP:SAMPLEPREP_SUMMARY cells. The extracts were scraped and mixed at 4°C for 15 min. After final SP:SAMPLEPREP_SUMMARY centrifugation at max speed for 15 min at 4°C, the supernatants were SP:SAMPLEPREP_SUMMARY transferred into LC-MS vials. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation of metabolites was achieved using a Millipore Sequant CH:CHROMATOGRAPHY_SUMMARY ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm CH:CHROMATOGRAPHY_SUMMARY guard column (both 5 mm particle size) with a binary solvent system. Solvent A CH:CHROMATOGRAPHY_SUMMARY was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was CH:CHROMATOGRAPHY_SUMMARY acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 CH:CHROMATOGRAPHY_SUMMARY °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 CH:CHROMATOGRAPHY_SUMMARY mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% CH:CHROMATOGRAPHY_SUMMARY B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B. CH:CHROMATOGRAPHY_SUMMARY Samples were randomized and the injection volume was 5 µl. A pooled quality CH:CHROMATOGRAPHY_SUMMARY control (QC) sample was generated from an equal mixture of all individual CH:CHROMATOGRAPHY_SUMMARY samples and analysed interspersed at regular intervals. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC-pHILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Metabolites were measured with a Thermo Scientific Q Exactive Hybrid MS:MS_COMMENTS Quadrupole-Orbitrap Mass spectrometer (HRMS) coupled to a Dionex Ultimate 3000 MS:MS_COMMENTS UHPLC. The mass spectrometer was operated in full-scan, polarity-switching mode, MS:MS_COMMENTS with the spray voltage set to +4.5 kV/-3.5 kV, the heated capillary held at 320 MS:MS_COMMENTS °C, and the auxiliary gas heater held at 280 °C. The sheath gas flow was set MS:MS_COMMENTS to 55 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow MS:MS_COMMENTS was set to 0 unit. HRMS data acquisition was performed in a range of m/z = MS:MS_COMMENTS 70–900, with the resolution set at 70,000, the AGC target at 1 × 106, and the MS:MS_COMMENTS maximum injection time (Max IT) at 120 ms. Metabolite identities were confirmed MS:MS_COMMENTS using two parameters: (1) precursor ion m/z was matched within 5 ppm of MS:MS_COMMENTS theoretical mass predicted by the chemical formula; (2) the retention time of MS:MS_COMMENTS metabolites was within 5% of the retention time of a purified standard run with MS:MS_COMMENTS the same chromatographic method. Chromatogram review and peak area integration MS:MS_COMMENTS were performed using the Thermo Fisher software Tracefinder 5.0 and the peak MS:MS_COMMENTS area for each detected metabolite was normalized against the total ion count MS:MS_COMMENTS (TIC) of that sample to correct any variations introduced from sample handling MS:MS_COMMENTS through instrument analysis. The normalized areas were used as variables for MS:MS_COMMENTS further statistical data analysis. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples MS48-01 MS48-02 MS48-03 MS48-04 MS48-05 MS48-06 MS48-07 MS48-08 MS48-09 MS48-10 MS49-11 MS48-12 MS48-13 MS48-14 MS48-15 MS48-16 MS48-17 MS48-18 MS48-19 MS48-20 Factors Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine Cell line:HK2 | Treatment:15N-leucine+15N-isoleucine Cell line:786-O | Treatment:15N-leucine+15N-isoleucine Cell line:786-O | Treatment:15N-leucine+15N-isoleucine Cell line:786-O | Treatment:15N-leucine+15N-isoleucine Cell line:786-O | Treatment:15N-leucine+15N-isoleucine Cell line:786-O | Treatment:15N-leucine+15N-isoleucine Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M1A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine Cell line:786-M2A | Treatment:15N-leucine+15N-isoleucine leucine+isoleucine_15N0 918991092.1 989710423.3 890084813.5 762139435.2 785235263.8 717869543.5 739991695 764887066.7 724782828 757228953.3 764243769 901937091.7 781105358.1 791086170.1 828938683.7 755923757.4 767757423.7 721210949.2 800343974.5 931421344.8 leucine+isoleucine_15N1 1004476089 1021164054 927712984.8 838282300.1 872321397.2 1168817726 1194203613 1116848353 894402565.5 1085909784 1395504197 1344742234 1283926288 1344045820 1240721024 1512212700 1380280190 1378235898 1503296835 1506811368 alanine_15N0 1228662844 1368355370 1350971919 1177990202 1189847469 543175808.3 510049431.6 517418972.3 464427833.7 545249008.3 563861130.6 576594000.2 539906390.5 543557199.1 540005082.5 539897251.3 531321348.1 528072781.6 551552384.7 623604887.9 alanine_15N1 130650132.4 132157360.1 125245705 111991768.7 119001938.6 27248658.95 30680862.56 29852884.75 25391443.8 32223063 31182184.84 40010802.38 33573921.74 36602923.68 37399426.34 37215298.42 42190704.45 38321454.7 41064728.57 48172680.94 arginine_15N0 61700569.19 127762879.8 228901368.9 64843619.1 32490279.88 21828237.97 12111725.97 20678799.03 254621285.8 33101658.05 42244725.51 37439102.85 53601985.1 30725438.24 36429546.41 50596882 43650998.26 25438660.65 59328247.53 92354087.13 arginine_15N1 958816.9068 1733506.684 3343086.491 934999.0233 269353.1018 54349.49208 0 132917.8389 3017695.754 173350.8375 1861708.531 1835119.459 2688243.208 1468630.894 1879717.152 2611747.263 2131934.568 1247852.97 2885488.146 4876863.553 argininosuccinate_15N0 1990292.963 2061535.92 1858160.206 1607188.075 1832865.889 0 0 0 0 0 1822040.591 2600874.221 1921183.281 1918739.132 2165947.26 2035820.433 2112802.255 1944611.102 2322822.333 2563910.131 argininosuccinate_15N1 288446.9914 275254.736 158825.4686 241237.7465 147595.2012 0 0 0 0 0 358840.4436 443007.0052 437309.7495 500933.8447 422700.7136 350056.5653 444438.8856 358486.6666 383005.1875 497563.3553 asparagine_15N0 112239780.5 114669051.1 101955796.1 92212858.75 96690994.57 54859609.2 55313914.73 55244079.27 43670180.37 54887345.23 59409534.6 63465177.13 59725178.4 60369596.34 60631659.1 64875500.55 64837496.93 60993782.15 66554808.37 73473553.32 asparagine_15N1 3063277.629 3148624.298 3040325.601 2724491.561 2788065.292 4939468.804 4886339.474 4587957.892 3138989.737 4667547.234 5147137.008 5676022.619 5263969.58 5524120.951 5561154.772 5868876.561 5889654.046 5330308.655 6079473.888 6560976.361 aspartate_15N0 968933754 949955437.1 1017212805 940857730.1 971702117.6 729029811.1 749403881.2 759141080.1 619958005.3 733017184.8 1024212460 1065770677 960487200.2 1033785545 1019256106 989324214.8 932315057.1 966734996.1 1036293377 1057433981 aspartate_15N1 216888797.5 227095765.1 228419572.8 210259581.4 219136518.3 212585102.2 216381285.1 210603092.2 164350738.1 203781031.1 281632890.9 282650021.3 254534776 276429460 265612840.5 258212997.3 238598240.1 247084053.8 268598530.7 266195710.5 glutamate_15N0 4133712591 4199878942 4279331702 3983172473 4113328124 4528880478 4647168081 4686086131 3995027591 4518198234 5208549566 5593776932 5132393699 5342677733 5287573956 5334713437 5198157852 5221788639 5563322400 5713770477 glutamate_15N1 954671265.6 962036503.5 974663979.8 903394688.2 935156989.3 1333523317 1357356066 1340900540 1145535711 1282631808 1451126215 1494951512 1387301338 1463703711 1398487739 1427253549 1347034205 1360912700 1463974760 1453856373 glutamine_15N0 203640827.1 218184957.2 240797816.6 193474529.7 193250812.7 576150903.3 582244195 533291731.6 392514436.3 519409944.5 597064944.1 529141794.8 531040560.8 559031715.8 479386802.1 629348485.4 543872934.1 552849437 592684709.1 562157649.8 glutamine_15N1 16678547.62 17130683.09 17089589.5 14756302.76 15112404.78 4178472.399 4104926.258 3758959.399 2963864.776 3697936.088 4438297.735 3792980.758 3844099.586 4108586.532 3352272.214 4533548.088 4060282.715 3890746.131 4221934.317 3953123.954 proline_15N0 24323520569 24366349412 23470997711 21886572258 22386205014 18563260179 18650071238 18779742324 15234559053 17738367124 19884751689 20289991164 20637689617 19734716162 19137596802 18646525339 18797401642 18530017081 19323968051 19883347915 proline_15N1 1489264840 1498984698 1495250295 1415970117 1471268043 2074074306 2083629239 2093067229 1618691702 1959990645 2252936861 2159452965 2358553925 2276797827 2135937794 1836901558 1829968311 1887605858 1948292341 1941188764 serine_15N0 86942852.64 86335552.24 80457314.54 73708096.87 76531081.89 22993939.49 22331694.05 22564390.44 48421826.8 25160257.48 24321890.87 24776964.35 24136777.87 23076952.12 23157576.88 26438494.42 24551329.14 23036849.41 25970666.17 28517905.86 serine_15N1 1696216.287 1499581.008 935112.0778 1281288.935 1111133.092 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB_ID MolecularFormula KEGG_ID Chemspider_ID Pubchem_ID CAS leucine+isoleucine_15N0 HMDB0000687 C6H13NO2 C00123 5880 6106 328-39-2 leucine+isoleucine_15N1 HMDB0000687 C6H13NO2 C00123 5880 6106 328-39-2 alanine_15N0 HMDB0000161 C3H7NO2 C00041 5735 5950 56-41-7 alanine_15N1 HMDB0000161 C3H7NO2 C00041 5735 5950 56-41-7 arginine_15N0 HMDB0000517 C6H14N4O2 C00062 6082 6322 1119-34-2 arginine_15N1 HMDB0000517 C6H14N4O2 C00062 6082 6322 1119-34-2 argininosuccinate_15N0 HMDB0000052 C10H18N4O6 C03406 16059 16950 2387-71-5 argininosuccinate_15N1 HMDB0000052 C10H18N4O6 C03406 16059 16950 2387-71-5 asparagine_15N0 HMDB0000168 C4H8N2O3 C00152 6031 6267 70-47-3 asparagine_15N1 HMDB0000168 C4H8N2O3 C00152 6031 6267 70-47-3 aspartate_15N0 HMDB0000191 C4H7NO4 C00049 5745 5960 56-84-8 aspartate_15N1 HMDB0000191 C4H7NO4 C00049 5745 5960 56-84-8 glutamate_15N0 HMDB0000148 C5H9NO4 C00025 NA 33032 56-86-0 glutamate_15N1 HMDB0000148 C5H9NO4 C00025 NA 33032 56-86-0 glutamine_15N0 HMDB0000641 C5H10N2O3 C00064 5746 5961 56-85-9 glutamine_15N1 HMDB0000641 C5H10N2O3 C00064 5746 5961 56-85-9 proline_15N0 HMDB0000162 C5H9NO2 C16435 128566 145742 147-85-3 proline_15N1 HMDB0000162 C5H9NO2 C16435 128566 145742 147-85-3 serine_15N0 HMDB0000187 C3H7NO3 C00065 5736 5951 56-45-1 serine_15N1 HMDB0000187 C3H7NO3 C00065 5736 5951 56-45-1 METABOLITES_END #END