#METABOLOMICS WORKBENCH zhouwei_20230523_194203 DATATRACK_ID:4039 STUDY_ID:ST002719 ANALYSIS_ID:AN004408 PROJECT_ID:PR001686 VERSION 1 CREATED_ON May 30, 2023, 1:43 pm #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 #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 (HIM), University of Chinese Academy of Sciences ST:INSTITUTE (Zhejiang Cancer Hospital), Chinese Academy of Sciences ST:LAST_NAME Zhou ST:FIRST_NAME Wei ST:ADDRESS Banshan Road ST:EMAIL zhouwei1989@iccas.ac.cn ST:PHONE 057188122431 #SUBJECT SU:SUBJECT_TYPE Cultured cells 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 - 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 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 #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 37 °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 ℃ for 20 min. Finally take the supernatant into the sample bottle for LC-MS/MS SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Nexera UHPLC LC-30A CH:COLUMN_NAME Merck SeQuant ZIC-pHILIC (100 x 2.1mm,5um) CH:SOLVENT_A 10mmol/L ammonium acetate+0.3% ammonia CH:SOLVENT_B 90% acetonitrile CH:FLOW_GRADIENT 0min A/B (5:95 V/V), 9.5min (50:50 V/V), 11.1 min（5:95 V/V）,14 min (5:95 CH:FLOW_GRADIENT V/V)) CH:FLOW_RATE 0.4 ml/min CH:COLUMN_TEMPERATURE 40℃ #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:ION_MODE POSITIVE 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 ∘C; 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_METABOLITE_DATA MS_METABOLITE_DATA:UNITS relative intensity MS_METABOLITE_DATA_START Samples A549-NC-1 A549-NC-2 A549-NC-3 A549-GAA-1 A549-GAA-2 A549-GAA-3 Factors Treatment:Control Treatment:Control Treatment:Control Treatment:10μM Treatment:10μM Treatment:10μM L-Asparagine 637650 624530 596150 598230 383120 436210 Succinate/Succinic acid 1553100 1229900 1661700 792440 440800 498360 L-Serine 882920 931770 907070 921810 627740 676180 L-Aspartate 5389000 5648900 5755600 2430500 1599200 1474400 L-Threonine 1070700 1210300 1183600 1044600 708930 783420 L-Alanine 9318.6 9269.5 11385 4559.9 3216.3 4586.9 Adenine 181210 168550 192310 41145 29161 30762 Inosine 78478000 58245000 83640000 6171500 23141000 14199000 Citrate/Citric acid 1023700 564520 863240 784750 947800 720330 L-Lactate 13030000 13988000 13742000 15127000 10778000 10679000 ADP 1116200 1088400 1005500 2027900 857780 915450 Fumarate/Fumaric acid 1082700 1283200 1285300 1162800 805050 728870 Uracil 614080 1211200 1063200 249720 128210 148140 Guanosine 3985200 3881000 3588900 157370 1012500 494650 Thiamine pyrophosphate (TPP) 30579 30237 22766 29665 19633 19232 Oxaloacetate 12873 27409 18960 13796 10907 11770 Methylmalonate 1643900 1330400 1767900 882270 479680 581770 Malate 8225900 8765600 10231000 8718700 5393400 4976400 Alpha-Ketoglutaric Acid 401910 468280 404890 253640 131730 111120 L-Glutamate 50273000 49687000 49114000 21729000 14855000 15238000 Aconitate 45720 20460 38827 27548 39218 24342 L-Tyrosine 831000 986420 636860 399910 253130 265520 Glyoxylate 51548 49163 37814 37810 42256 30819 NAD+ 644960 469290 348800 275340 196390 257080 D-Glucose 6-phosphate 94419 49434 198790 513200 640170 680230 Fructose 1,6-bisphosphate 851170 1242900 1710000 8443000 11940000 16383000 D-Fructose 6-phosphate 9 9 19008 55026 64787 72789 D-Glucose 1-phosphate 310020 284220 415670 551880 338410 387790 Glycerol 3-phosphate 640110 687040 555360 5538100 3261900 4114800 Phosphoenolpyruvic acid 14858 23460 24646 81233 41951 47631 6-Phosphogluconic acid 178680 9 300390 591890 1215600 1101500 Pyruvic acid 29771 30623 38272 31679 22848 29653 D-Erythrose 4-phosphate 9 9 9 28396 29885 36339 sedoheptulose 7-phosphate 1315000 872150 1134600 801630 441200 583810 D-Ribulose 5-phosphate 5731400 6429400 8062600 2553700 3400700 3151800 Dihydroxyacetone phosphate 917550 1094400 1001000 9 9 9 isocitric acid 158550 67153 190360 36853 20457 18637 AMP 12948000 17501000 16950000 429040 1702600 1566800 GMP 399050 600740 591380 9954.5 38818 31211 dCMP 21825 14964 16527 9 9 9 dAMP 20696 30943 30669 9 9 9 IMP 531920 692480 538090 330850 268650 347220 UMP 2191300 2965500 2656900 57564 201120 191810 dTMP 108490 190270 164690 9 9 9 dUMP 16155 38830 27360 9 9 9 D-Glutamine 1167500 987280 1003800 1674200 1320100 1323600 PRPP 39951 40438 51093 497960 276180 330960 UDP-GlcNAc 16249000 13927000 17011000 17194000 12082000 10557000 BPG 244120 321310 322320 1208100 565690 752540 ATP 13084000 11742000 10273000 27189000 10027000 10992000 2-Phospho-D-glyceric acid 88382 103280 102280 633130 598050 621060 Guanosine diphosphate (GDP) 691290 537990 579300 175250 230310 242550 Glyceraldehyde 3-phosphate 685170 727130 789170 9 9 9 Xyluose-5-phosphate 1695300 1792100 2497500 1606700 1950100 1927500 argininosuccinic acid 40668 45214 38945 36237 30140 26413 phosphorylethanolamine 62215 62701 72366 4578100 3811500 3437100 L-citrulline 113560 85819 111810 103870 76230 92181 L-Arginine 21584000 23096000 23614000 22195000 16115000 17797000 L-Cystine 52497 76395 69469 32666 10062 8473.4 Acetyl-CoA 584720 506870 566180 763210 165740 274860 Succinyl-CoA 10128 24104 17538 11235 9 7833.6 Beta-Leucine 20444000 21445000 21411000 18333000 13536000 14359000 Lysine 22515000 21219000 20465000 33248000 29571000 30770000 L-cysteine 3497.8 4006.7 5057.6 3176.3 2788.7 2756.8 ornithine 48565 53402 59503 97094 67398 80708 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG CID HMDB Pubchem CID CAS ChEBI Metlin L-Asparagine C00152 HMDB00168 6267 70-47-3 17196 14 Succinate/Succinic acid C00042 HMDB00254 1110 110-15-6 - - L-Serine C00065 HMDB00187 5951 56-45-1 17115 5203 L-Aspartate C00049 HMDB00191 5960 56-84-8 17053 5206 L-Threonine C00188 HMDB00167 6288 72-19-5 16857 32 L-Alanine C00041 HMDB00161 5950 56-41-7 16977 - Adenine C00147 HMDB00034 190 73-24-5 16708 85 Inosine C00294 HMDB00195 6021 58-63-9 17596 84 Citrate/Citric acid C00158 HMDB00094 311 77-92-9 30769 124 L-Lactate C00186 HMDB00190 107689 79-33-4 422 5205 ADP C00008 HMDB01341 6022 58-64-0 16761 6175 Fumarate/Fumaric acid C00122 HMDB00134 444972 110-17-8 18012 3242 Uracil C00106 HMDB00300 1174 66-22-8 17568 258 Guanosine C00387 HMDB00133 6802 118-00-3 16750 87 Thiamine pyrophosphate (TPP) C00068 HMDB01372 1132 154-87-0 9532 2832 Oxaloacetate C00036 HMDB00223 970 328-42-7 30744 123 Methylmalonate C02170 HMDB00202 487 516-05-2 30860 3712 Malate C00149 HMDB00744 525 6915-15-7 6650 118 Alpha-Ketoglutaric Acid C00026 HMDB00208 51 328-50-7 30915 5218 L-Glutamate - HMDB0003339 23327 6893-26-1 15966 6895 Aconitate C00417 HMDB00072 643757 585-84-2 32805 5130 L-Tyrosine C00082 HMDB00158 6057 60-18-4 17895 34 Glyoxylate C00048 HMDB0000119 760 298-12-4 16891 3213 NAD+ C00003 HMDB00902 5893 53-84-9 15846 5858 D-Glucose 6-phosphate C00092 HMDB01401 439958 56-73-5 4170 145 Fructose 1,6-bisphosphate - HMDB0001058 10267 34693-15-7 28013 147 D-Fructose 6-phosphate C00085 HMDB0000124 69507 643-13-0 15946 5159 D-Glucose 1-phosphate C00103 HMDB0001586 439165 59-56-3 16077 6331 Glycerol 3-phosphate C00093 HMDB0000126 439162 57-03-4 15978 5161 Phosphoenolpyruvic acid C00074 HMDB0000263 1005 138-08-9 - - 6-Phosphogluconic acid - HMDB0001316 91493 921-62-0 48928 367 Pyruvic acid C00022 HMDB00243 1060 127-17-3 - - D-Erythrose 4-phosphate C00279 HMDB0001321 122357 585-18-2 48153 6158 sedoheptulose 7-phosphate C00281 HMDB0001068 22833559 2646-35-7 15721 - D-Ribulose 5-phosphate - HMDB0000618 439184 4151-19-3 17363 - Dihydroxyacetone phosphate C00111 HMDB0001473 668 57-04-5 16108 6262 isocitric acid C00311 HMDB0000193 1198 320-77-4 30887 3328 AMP C00020 HMDB00045 6083 61-19-8 16027 5111 GMP C00144 HMDB01397 6804 85-32-5 17345 6216 dCMP C00239 HMDB01202 13945 1032-65-1 15918 6078 dAMP C00360 HMDB00905 12599 653-63-4 17713 3461 IMP C00130 HMDB00175 8582 131-99-7 17202 5196 UMP C00105 HMDB00288 6030 58-97-9 16695 - dTMP C00364 HMDB01227 9700 365-07-1 17013 6092 dUMP C00365 HMDB0001409 65063 964-26-1 17622 6225 D-Glutamine C00819 HMDB0003423 145815 5959-95-5 17061 6926 PRPP C00119 HMDB0000280 7339 7540-64-9 17111 5274 UDP-GlcNAc C00043 HMDB0000290 445675 528-04-1 16264 5281 BPG C03339 HMDB0001294 186004 138-81-8 17720 153 ATP - HMDB0000538 5957 56-65-5 15422 5523 2-Phospho-D-glyceric acid - HMDB0003391 439278 3443-57-0 17835 6915 Guanosine diphosphate (GDP) C00035 HMDB0001201 8977 146-91-8 17552 6077 Glyceraldehyde 3-phosphate - HMDB0001112 729 142-10-9 17138 3294 Xyluose-5-phosphate - HMDB0000868 439190 4212-65-1 16332 5829 argininosuccinic acid C03406 HMDB0000052 16950 2387-71-5 15682 5115 phosphorylethanolamine C00346 HMDB0000224 1015 1071-23-4 17553 5233 L-citrulline C00327 HMDB0000904 9750 372-75-8 16349 16 L-Arginine C00062 HMDB00517 6322 74-79-3 16467 5502 L-Cystine C00491 HMDB00192 67678 56-89-3 16283 5207 Acetyl-CoA C00024 HMDB0001206 444493 72-89-9 15351 6082 Succinyl-CoA C00091 HMDB0001022 439161 604-98-8 15380 5951 Beta-Leucine C02486 HMDB0003640 439734 5699-54-7 86261 - Lysine C00047 HMDB0000182 5962 56-87-1 18019 5200 L-cysteine C00097 HMDB0000574 5862 52-90-4 17561 5556 ornithine C00077 HMDB0000214 76654 3184-13-2 15729 27 METABOLITES_END #END