#METABOLOMICS WORKBENCH zhouwei_20230523_194203 DATATRACK_ID:4039 STUDY_ID:ST002719 ANALYSIS_ID:AN004408 PROJECT_ID:PR001686 VERSION 1 CREATED_ON 01-17-2025 #PROJECT PR:PROJECT_TITLE Comparison of metabolic of A549 cells before and after Gossypol acetate (GAA) PR:PROJECT_TITLE treatment PR:PROJECT_SUMMARY A549 cells were seeded into 6-well cell culture dishes and treated with DMSO or PR:PROJECT_SUMMARY 10 μM GAA for 48 h. Analysis of energy metabolites using liquid PR:PROJECT_SUMMARY chromatography-mass spectrometry. PR:INSTITUTE Hangzhou Institute of Medicine (HIM), University of Chinese Academy of Sciences PR:INSTITUTE (Zhejiang Cancer Hospital), Chinese Academy of Sciences PR:LAST_NAME Zhou PR:FIRST_NAME Wei PR:ADDRESS Banshan Road PR:EMAIL zhouwei1989@iccas.ac.cn PR:PHONE 057188122431 PR:DOI http://dx.doi.org/10.21228/M8S13H #STUDY ST:STUDY_TITLE Comparison of metabolic of A549 cells before and after Gossypol acetate (GAA) ST:STUDY_TITLE treatment ST:STUDY_SUMMARY GAA is a natural product with anti-cancer application prospect, but its ST:STUDY_SUMMARY anti-tumor molecular mechanism is still controversial. Previous studies have ST:STUDY_SUMMARY showed that GAA can suppress the expression of mitochondrial DNA encoded ST:STUDY_SUMMARY proteins by targeting LRPPRC, suggesting that GAA may affect mitochondrial ST:STUDY_SUMMARY metabolism. Here, metabonomics was applied to study of effect of GAA on central ST:STUDY_SUMMARY carbon metabolism in A549 cells. The metabolomic data showed that GAA ST:STUDY_SUMMARY significantly decreased tricarboxylic acid cycle ralated metabolites and ST:STUDY_SUMMARY significantly increased glycolysis-related metabolites. These results indicated ST:STUDY_SUMMARY that GAA could inhibite oxidative phosphorylation in A549 cells. ST:INSTITUTE Hangzhou Institute of Medicine, Chinese Academy of Sciences ST:LABORATORY University of Chinese Academy of Sciences (Zhejiang Cancer Hospital) ST:LAST_NAME Zhou ST:FIRST_NAME Wei ST:ADDRESS Banshan Road ST:EMAIL zhouwei1989@iccas.ac.cn ST:PHONE 057188122431 ST:SUBMIT_DATE 2023-05-23 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:SPECIES_GROUP Mammals #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - A549-GAA-1 Treatment:10μM RAW_FILE_NAME=A549-GAA-1.mzML SUBJECT_SAMPLE_FACTORS - A549-GAA-2 Treatment:10μM RAW_FILE_NAME=A549-GAA-2.mzML SUBJECT_SAMPLE_FACTORS - A549-GAA-3 Treatment:10μM RAW_FILE_NAME=A549-GAA-3.mzML SUBJECT_SAMPLE_FACTORS - A549-NC-1 Treatment:Control RAW_FILE_NAME=A549-NC-1.mzML SUBJECT_SAMPLE_FACTORS - A549-NC-2 Treatment:Control RAW_FILE_NAME=A549-NC-2.mzML SUBJECT_SAMPLE_FACTORS - A549-NC-3 Treatment:Control RAW_FILE_NAME=A549-NC-3.mzML #COLLECTION CO:COLLECTION_SUMMARY Cultured cells were collected by trypsin digestion and low-speed centrifugation. CO:COLLECTION_SUMMARY The collected cells were rapidly cooled using liquid nitrogen, and the cooled CO:COLLECTION_SUMMARY samples were stored in a cryogenic refrigerator at -80 degrees Celsius. CO:SAMPLE_TYPE Lung #TREATMENT TR:TREATMENT_SUMMARY A549 cells were seeded in six-well plates and cultured at 37â°C and 5% CO2. TR:TREATMENT_SUMMARY When the cell confluence reached about 60%, the corresponding final TR:TREATMENT_SUMMARY concentration of GAA or equivalent volume of DMSO was added. The final volume of TR:TREATMENT_SUMMARY the medium was 3 ml/well, and the treatment time was 48 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The collected cells were thawed on ice, then added 500 μM pre-cooled extractant SP:SAMPLEPREP_SUMMARY (80% methanol aqueous solution), and whirl for 2 min. Freeze the mixture for 5 SP:SAMPLEPREP_SUMMARY min in liquid nitrogen after remove ice for 5 min, it will be whirled for 2 min, SP:SAMPLEPREP_SUMMARY circulate this at 3 times. Centrifuge the mixture again with 15000 rpm/min at 4 SP:SAMPLEPREP_SUMMARY â for 20 min. Finally take the supernatant into the sample bottle for LC-MS/MS SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Nexera UHPLC LC-30A CH:COLUMN_NAME Merck SeQuant ZIC-pHILIC (100 x 2.1mm,5um) CH:COLUMN_TEMPERATURE 40â CH:FLOW_GRADIENT 0min A/B (5:95 V/V), 9.5min (50:50 V/V), 11.1 minï¼5:95 V/Vï¼,14 min (5:95 CH:FLOW_GRADIENT V/V)) CH:FLOW_RATE 0.4 ml/min CH:SOLVENT_A 10mmol/L ammonium acetate+0.3% ammonia CH:SOLVENT_B 90% acetonitrile CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Protocol.pdf #MS MS:INSTRUMENT_NAME ABI Sciex API 6500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS The data collection system mainly includes ultra-high performance liquid MS:MS_COMMENTS chromatography and tandem mass spectrometry. LIT and triple quadrupole (QQQ) MS:MS_COMMENTS scans were acquired on a triple quadrupole-linear ion trap mass MS:MS_COMMENTS spectrometer(QTRAP), QTRAP® LC-MS/MS System, equipped with an ESI Turbo MS:MS_COMMENTS Ion-Spray interface, operatingin positive and negative ion mode and controlled MS:MS_COMMENTS by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as MS:MS_COMMENTS follows: source temperature 450 âC; ion spray voltage (IS) 5500 V (positive), MS:MS_COMMENTS -4500 V (negative); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) MS:MS_COMMENTS were set at 40, 55, and 35.0 psi, respectively; the collision gas (CAD) was MS:MS_COMMENTS medium. Instrument tuning and mass calibration were performed with 10 and 100 MS:MS_COMMENTS μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. A MS:MS_COMMENTS specific set of MRM transitions were monitored for each period according to the MS:MS_COMMENTS metabolites eluted within this period. MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS relative intensity MS_METABOLITE_DATA_START Samples A549-GAA-1 A549-GAA-2 A549-GAA-3 A549-NC-1 A549-NC-2 A549-NC-3 Factors Treatment:10μM Treatment:10μM Treatment:10μM Treatment:Control Treatment:Control Treatment:Control 2-Phospho-D-glyceric acid 633130.0000 598050.0000 621060.0000 88382.0000 103280.0000 102280.0000 6-Phosphogluconic acid 591890.0000 1215600.0000 1101500.0000 178680.0000 9.0000 300390.0000 Acetyl-CoA 763210.0000 165740.0000 274860.0000 584720.0000 506870.0000 566180.0000 Aconitate 27548.0000 39218.0000 24342.0000 45720.0000 20460.0000 38827.0000 Adenine 41145.0000 29161.0000 30762.0000 181210.0000 168550.0000 192310.0000 ADP 2027900.0000 857780.0000 915450.0000 1116200.0000 1088400.0000 1005500.0000 Alpha-Ketoglutaric Acid 253640.0000 131730.0000 111120.0000 401910.0000 468280.0000 404890.0000 AMP 429040.0000 1702600.0000 1566800.0000 12948000.0000 17501000.0000 16950000.0000 argininosuccinic acid 36237.0000 30140.0000 26413.0000 40668.0000 45214.0000 38945.0000 ATP 27189000.0000 10027000.0000 10992000.0000 13084000.0000 11742000.0000 10273000.0000 Beta-Leucine 18333000.0000 13536000.0000 14359000.0000 20444000.0000 21445000.0000 21411000.0000 BPG 1208100.0000 565690.0000 752540.0000 244120.0000 321310.0000 322320.0000 Citrate/Citric acid 784750.0000 947800.0000 720330.0000 1023700.0000 564520.0000 863240.0000 dAMP 9.0000 9.0000 9.0000 20696.0000 30943.0000 30669.0000 dCMP 9.0000 9.0000 9.0000 21825.0000 14964.0000 16527.0000 D-Erythrose 4-phosphate 28396.0000 29885.0000 36339.0000 9.0000 9.0000 9.0000 D-Fructose 6-phosphate 55026.0000 64787.0000 72789.0000 9.0000 9.0000 19008.0000 D-Glucose 1-phosphate 551880.0000 338410.0000 387790.0000 310020.0000 284220.0000 415670.0000 D-Glucose 6-phosphate 513200.0000 640170.0000 680230.0000 94419.0000 49434.0000 198790.0000 D-Glutamine 1674200.0000 1320100.0000 1323600.0000 1167500.0000 987280.0000 1003800.0000 Dihydroxyacetone phosphate 9.0000 9.0000 9.0000 917550.0000 1094400.0000 1001000.0000 D-Ribulose 5-phosphate 2553700.0000 3400700.0000 3151800.0000 5731400.0000 6429400.0000 8062600.0000 dTMP 9.0000 9.0000 9.0000 108490.0000 190270.0000 164690.0000 dUMP 9.0000 9.0000 9.0000 16155.0000 38830.0000 27360.0000 Fructose 1_6-bisphosphate 8443000.0000 11940000.0000 16383000.0000 851170.0000 1242900.0000 1710000.0000 Fumarate/Fumaric acid 1162800.0000 805050.0000 728870.0000 1082700.0000 1283200.0000 1285300.0000 Glyceraldehyde 3-phosphate 9.0000 9.0000 9.0000 685170.0000 727130.0000 789170.0000 Glycerol 3-phosphate 5538100.0000 3261900.0000 4114800.0000 640110.0000 687040.0000 555360.0000 Glyoxylate 37810.0000 42256.0000 30819.0000 51548.0000 49163.0000 37814.0000 GMP 9954.5000 38818.0000 31211.0000 399050.0000 600740.0000 591380.0000 Guanosine 157370.0000 1012500.0000 494650.0000 3985200.0000 3881000.0000 3588900.0000 Guanosine diphosphate (GDP) 175250.0000 230310.0000 242550.0000 691290.0000 537990.0000 579300.0000 IMP 330850.0000 268650.0000 347220.0000 531920.0000 692480.0000 538090.0000 Inosine 6171500.0000 23141000.0000 14199000.0000 78478000.0000 58245000.0000 83640000.0000 isocitric acid 36853.0000 20457.0000 18637.0000 158550.0000 67153.0000 190360.0000 L-Alanine 4559.9000 3216.3000 4586.9000 9318.6000 9269.5000 11385.0000 L-Arginine 22195000.0000 16115000.0000 17797000.0000 21584000.0000 23096000.0000 23614000.0000 L-Asparagine 598230.0000 383120.0000 436210.0000 637650.0000 624530.0000 596150.0000 L-Aspartate 2430500.0000 1599200.0000 1474400.0000 5389000.0000 5648900.0000 5755600.0000 L-citrulline 103870.0000 76230.0000 92181.0000 113560.0000 85819.0000 111810.0000 L-cysteine 3176.3000 2788.7000 2756.8000 3497.8000 4006.7000 5057.6000 L-Cystine 32666.0000 10062.0000 8473.4000 52497.0000 76395.0000 69469.0000 L-Glutamate 21729000.0000 14855000.0000 15238000.0000 50273000.0000 49687000.0000 49114000.0000 L-Lactate 15127000.0000 10778000.0000 10679000.0000 13030000.0000 13988000.0000 13742000.0000 L-Serine 921810.0000 627740.0000 676180.0000 882920.0000 931770.0000 907070.0000 L-Threonine 1044600.0000 708930.0000 783420.0000 1070700.0000 1210300.0000 1183600.0000 L-Tyrosine 399910.0000 253130.0000 265520.0000 831000.0000 986420.0000 636860.0000 Lysine 33248000.0000 29571000.0000 30770000.0000 22515000.0000 21219000.0000 20465000.0000 Malate 8718700.0000 5393400.0000 4976400.0000 8225900.0000 8765600.0000 10231000.0000 Methylmalonate 882270.0000 479680.0000 581770.0000 1643900.0000 1330400.0000 1767900.0000 NAD+ 275340.0000 196390.0000 257080.0000 644960.0000 469290.0000 348800.0000 ornithine 97094.0000 67398.0000 80708.0000 48565.0000 53402.0000 59503.0000 Oxaloacetate 13796.0000 10907.0000 11770.0000 12873.0000 27409.0000 18960.0000 Phosphoenolpyruvic acid 81233.0000 41951.0000 47631.0000 14858.0000 23460.0000 24646.0000 phosphorylethanolamine 4578100.0000 3811500.0000 3437100.0000 62215.0000 62701.0000 72366.0000 PRPP 497960.0000 276180.0000 330960.0000 39951.0000 40438.0000 51093.0000 Pyruvic acid 31679.0000 22848.0000 29653.0000 29771.0000 30623.0000 38272.0000 sedoheptulose 7-phosphate 801630.0000 441200.0000 583810.0000 1315000.0000 872150.0000 1134600.0000 Succinate/Succinic acid 792440.0000 440800.0000 498360.0000 1553100.0000 1229900.0000 1661700.0000 Succinyl-CoA 11235.0000 9.0000 7833.6000 10128.0000 24104.0000 17538.0000 Thiamine pyrophosphate (TPP) 29665.0000 19633.0000 19232.0000 30579.0000 30237.0000 22766.0000 UDP-GlcNAc 17194000.0000 12082000.0000 10557000.0000 16249000.0000 13927000.0000 17011000.0000 UMP 57564.0000 201120.0000 191810.0000 2191300.0000 2965500.0000 2656900.0000 Uracil 249720.0000 128210.0000 148140.0000 614080.0000 1211200.0000 1063200.0000 Xyluose-5-phosphate 1606700.0000 1950100.0000 1927500.0000 1695300.0000 1792100.0000 2497500.0000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant 2-Phospho-D-glyceric acid 439278 6-Phosphogluconic acid 91493 Acetyl-CoA 444493 C00024 Aconitate 643757 C00417 Adenine 190 C00147 ADP 6022 C00008 Alpha-Ketoglutaric Acid 51 C00026 AMP 6083 C00020 argininosuccinic acid 16950 C03406 ATP 5957 Beta-Leucine 439734 C02486 BPG 186004 C03339 Citrate/Citric acid 311 C00158 dAMP 12599 C00360 dCMP 13945 C00239 D-Erythrose 4-phosphate 122357 C00279 D-Fructose 6-phosphate 69507 C00085 D-Glucose 1-phosphate 439165 C00103 D-Glucose 6-phosphate 439958 C00092 D-Glutamine 145815 C00819 Dihydroxyacetone phosphate 668 C00111 D-Ribulose 5-phosphate 439184 dTMP 9700 C00364 dUMP 65063 C00365 Fructose 1,6-bisphosphate 10267 Fumarate/Fumaric acid 444972 C00122 Glyceraldehyde 3-phosphate 729 Glycerol 3-phosphate 439162 C00093 Glyoxylate 760 C00048 GMP 6804 C00144 Guanosine 6802 C00387 Guanosine diphosphate (GDP) 8977 C00035 IMP 8582 C00130 Inosine 6021 C00294 isocitric acid 1198 C00311 L-Alanine 5950 C00041 L-Arginine 6322 C00062 L-Asparagine 6267 C00152 L-Aspartate 5960 C00049 L-citrulline 9750 C00327 L-cysteine 5862 C00097 L-Cystine 67678 C00491 L-Glutamate 23327 L-Lactate 107689 C00186 L-Serine 5951 C00065 L-Threonine 6288 C00188 L-Tyrosine 6057 C00082 Lysine 5962 C00047 Malate 525 C00149 Methylmalonate 487 C02170 NAD+ 5893 C00003 ornithine 76654 C00077 Oxaloacetate 970 C00036 Phosphoenolpyruvic acid 1005 C00074 phosphorylethanolamine 1015 C00346 PRPP 7339 C00119 Pyruvic acid 1060 C00022 sedoheptulose 7-phosphate 22833559 C00281 Succinate/Succinic acid 1110 C00042 Succinyl-CoA 439161 C00091 Thiamine pyrophosphate (TPP) 1132 C00068 UDP-GlcNAc 445675 C00043 UMP 6030 C00105 Uracil 1174 C00106 Xyluose-5-phosphate 439190 METABOLITES_END #END