#METABOLOMICS WORKBENCH OGDH_20240711_235807 DATATRACK_ID:5000 STUDY_ID:ST003345 ANALYSIS_ID:AN005480 PROJECT_ID:PR002081 VERSION 1 CREATED_ON July 24, 2024, 5:01 pm #PROJECT PR:PROJECT_TITLE OGDH (2-oxoglutarate dehydrogenase complex component E1) knockout A375 cells' PR:PROJECT_TITLE succinyl-CoA level upon glucose starvation PR:PROJECT_SUMMARY The adaptation of tumor cells to metabolic stress is crucial for tumor PR:PROJECT_SUMMARY development. We demonstrate that the translation of NRF2 (Nuclear factor PR:PROJECT_SUMMARY erythroid 2-related factor 2), a master regulator of antioxidant response, is PR:PROJECT_SUMMARY controlled by the OGDH (2-oxoglutarate dehydrogenase complex component PR:PROJECT_SUMMARY E1)-METTL3 (N6-adenosine-methyltransferase catalytic subunit) axis in melanoma PR:PROJECT_SUMMARY for glucose starvation adaptation. To elucidate the regulatory mechanism of the PR:PROJECT_SUMMARY OGDH-METTL3 axis during glucose starvation, we conducted co-immunoprecipitation PR:PROJECT_SUMMARY and mass spectrometry analysis to identify the binding partner of OGDH that PR:PROJECT_SUMMARY governs both the OGDH-METTL3 axis and tumor cell adaptation to glucose PR:PROJECT_SUMMARY deprivation. PR:INSTITUTE Xiamen University PR:LAST_NAME Xu PR:FIRST_NAME Kejun PR:ADDRESS Xiangshan Street 4221, Xiangan District, Xiamen, Fujian, 361100, China PR:EMAIL 1329490903@qq.com PR:PHONE +0086 150-6180-5973 #STUDY ST:STUDY_TITLE OGDH (2-oxoglutarate dehydrogenase complex component E1) knockout A375 cells' ST:STUDY_TITLE succinyl-CoA level upon glucose starvation ST:STUDY_SUMMARY The adaptation of tumor cells to metabolic stress is crucial for tumor ST:STUDY_SUMMARY development. We demonstrate that the translation of NRF2, a master regulator of ST:STUDY_SUMMARY antioxidant response, is controlled by the OGDH-METTL3 axis in melanoma for ST:STUDY_SUMMARY glucose starvation adaptation. To elucidate the regulatory mechanism of the ST:STUDY_SUMMARY OGDH-METTL3 axis during glucose starvation, we conducted co-immunoprecipitation ST:STUDY_SUMMARY and mass spectrometry analysis to identify the binding partner of OGDH that ST:STUDY_SUMMARY governs both the OGDH-METTL3 axis and tumor cell adaptation to glucose ST:STUDY_SUMMARY deprivation by detecting succinyl-CoA in total cells or cytoplasm. ST:INSTITUTE Xiamen University ST:LAST_NAME Xu ST:FIRST_NAME Kejun ST:ADDRESS Xiangshan Street 4221, Xiangan District, Xiamen, Fujian, 361100, China ST:EMAIL 1329490903@qq.com ST:PHONE 15061805973 #SUBJECT SU:SUBJECT_TYPE Human 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 1 SC_0 Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 2 SC_10nM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 3 SC_50nM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 4 SC_250nM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 5 SC_1.25uM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 6 SC_5uM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 7 SC_10uM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 8 SC_25uM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 9 SC_40nM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 10 SC_4uM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 11 SC_100nM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 12 SC_400nM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 13 SC_1uM Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 14 SC_10uM-2 Sample source:standard | Genotype:- Treatment=control; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 15 N1_1 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 16 N1_2 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 17 N1_3 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 18 N1_4 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 19 N1_5 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 20 N1_6 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 21 N1_7 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 22 N1_8 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 23 N1_9 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 24 N1_10 Sample source:A375 melanoma cells | Genotype:wild type A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 25 O5_1 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 26 O5_2 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 27 O5_3 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 28 O5_4 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 29 O5_5 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 30 O5_6 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 31 O5_7 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 32 O5_8 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 33 O5_9 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff SUBJECT_SAMPLE_FACTORS 34 O5_10 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Treatment=glucose starvation; RAW_FILE_NAME=Succinyl-CoA_CXY.wiff; RAW_FILE_NAME=Succinyl-CoA_CXY_Ac.wiff #COLLECTION CO:COLLECTION_SUMMARY Cells were harvested after glucose starvation for 3 h. Freeze by liquid nitrogen CO:COLLECTION_SUMMARY and 1 mL extraction liquid (Vmethanol: Vacetonitrile: Vwater = 2:2:1) was added. CO:COLLECTION_SUMMARY Vortex mixing for 30 s and incubate at -20 ℃ for 1 hour, then centrifuged for CO:COLLECTION_SUMMARY 15 min at 15000 g. The supernatant was transferred into a 1.5 mL tubes and dried CO:COLLECTION_SUMMARY at a gentle nitrogen flow. The dry metabolites were reconstituted by adding 100 CO:COLLECTION_SUMMARY μL extraction liquid (Vacetonitrile: Vwater = 1:1). After centrifugation for 15 CO:COLLECTION_SUMMARY min at 13000 g, supernatant was transfer to a fresh 2 mL LC/MS glass vial for CO:COLLECTION_SUMMARY the UHPLC-QQQ-MS analysis. To determine the levels of succinyl-CoA in cytosol. CO:COLLECTION_SUMMARY Cells were harvested after glucose starvation for 3 h. The cells were scratched CO:COLLECTION_SUMMARY from the dish, collected in ice-cold PBS, and washed twice with ice-cold PBS. CO:COLLECTION_SUMMARY Then, the cells were resuspended in 500 μL of digitonin lysis buffer (75 mM CO:COLLECTION_SUMMARY NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose, and 190 mg/mL digitonin). CO:COLLECTION_SUMMARY After 2 min on ice, the cells were spun for 30 min at 20,000 g at 4°C. Ten per CO:COLLECTION_SUMMARY cent trichloroacetic acid (TCA) in water was applied to remove proteins from the CO:COLLECTION_SUMMARY cytosol samples. The cytosol samples were transfer to a fresh 2 mL LC/MS glass CO:COLLECTION_SUMMARY vial for the UHPLC-QQQ-MS analysis. Cell volume was quantified by CM-H2DCFDA CO:COLLECTION_SUMMARY (Thermo Fisher Scientific, 2600176)and DAPI (Thermo Fisher Scientific, 62247). CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Prepare 100 mm or 60 mm culture dish of A375 Cells with 80%-90% density. The TR:TREATMENT_SUMMARY plates were then washed with DMEM without glucose firstly to remove residual TR:TREATMENT_SUMMARY DMEM containing glucose. Then we added DMEM without glucose again to starve TR:TREATMENT_SUMMARY cells without glucose for 3 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Vortex mixing for 30 s and incubate at -20 ℃ for 1 hour, then centrifuged for SP:SAMPLEPREP_SUMMARY 15 min at 15000 g. The supernatant was transferred into a 1.5 mL tubes and dried SP:SAMPLEPREP_SUMMARY at a gentle nitrogen flow. The cells were scratched from the dish, collected in SP:SAMPLEPREP_SUMMARY ice-cold PBS, and washed twice with ice-cold PBS. Then, the cells were SP:SAMPLEPREP_SUMMARY resuspended in 500 μL of digitonin lysis buffer (75 mM NaCl, 1 mM NaH2PO4, 8 mM SP:SAMPLEPREP_SUMMARY Na2HPO4, 250 mM sucrose, and 190 mg/mL digitonin). After 2 min on ice, the cells SP:SAMPLEPREP_SUMMARY were spun for 30 min at 20,000 g at 4°C. Ten per cent trichloroacetic acid SP:SAMPLEPREP_SUMMARY (TCA) in water was applied to remove proteins from the cytosol samples. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Millipore ZIC-pHILIC (100 × 2.1mm, 5um) CH:SOLVENT_A 100% water; 15mM ammonium acetate; 3 ml/L ammonium hydroxide (The content of CH:SOLVENT_A ammonium hydroxide in the ammonium hydroxide solution is greater than 28%) CH:SOLVENT_B 90% acetonitrile/10% water CH:FLOW_GRADIENT 95% B held for 2 min, decreased to 45% B in 13 min, held for 3 min, and the post CH:FLOW_GRADIENT time was set 4 min CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 40°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The QTRAP 5500 mass spectrometer (AB SCIEX) using an Turbo V ion source. The ion MS:MS_COMMENTS source was run in positive mode with a spray voltage of 5500 V, Gas 55 psi and MS:MS_COMMENTS Curtain gas 35 psi, Metabolites were measured using the multiple reactions MS:MS_COMMENTS monitoring mode (MRM) optimized using analytical standards, The amounts of MS:MS_COMMENTS metabolite were analysed by MultiQuant Software (AB SCIEX). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Calculated Concentration (μM) MS_METABOLITE_DATA_START Samples SC_0 SC_10nM SC_50nM SC_250nM SC_1.25uM SC_5uM SC_10uM SC_25uM SC_40nM SC_4uM SC_100nM SC_400nM SC_1uM SC_10uM-2 N1_1 N1_2 N1_3 N1_4 N1_5 N1_6 N1_7 N1_8 N1_9 N1_10 O5_1 O5_2 O5_3 O5_4 O5_5 O5_6 O5_7 O5_8 O5_9 O5_10 Factors Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:standard | Genotype:- Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:wild type A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 Sample source:A375 melanoma cells | Genotype:OGDH KO A375 succinyl-CoA N/A N/A N/A 0.252906539 1.192893944 4.452714041 11.03234331 26.00703876 37.96082457 4463.583375 113.2896083 410.9003934 856.5874947 9183.489809 2.551041264 1.833379495 1.524949969 1.560554529 1.572024938 85.39344141 69.65879689 65.46056547 39.70635731 82.09646078 0.736605227 0.501600362 0.74329633 0.644538088 2.436629571 55.15375457 53.78023198 66.28333665 62.34909005 60.41509583 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem_ID KEGG_ID succinyl-CoA 92133 C00091 METABOLITES_END #END