#METABOLOMICS WORKBENCH Andre_Gollowitzer_20240709_115832 DATATRACK_ID:4993 STUDY_ID:ST003337 ANALYSIS_ID:AN005469 PROJECT_ID:PR002065 VERSION 1 CREATED_ON July 22, 2024, 6:00 pm #PROJECT PR:PROJECT_TITLE Changes in phospholipid fatty acid composition under cytotoxic stress facilitate PR:PROJECT_TITLE peroxidation PR:PROJECT_SUMMARY Programs leading to cell death, such as apoptosis, necroptosis, and ferroptosis, PR:PROJECT_SUMMARY involve an oxidative component linked to lipid metabolism that influences PR:PROJECT_SUMMARY membrane homeostasis. Emerging evidence suggests inter-program cross-talk, PR:PROJECT_SUMMARY emphasizing the need for overarching regulatory mechanisms. We show that under PR:PROJECT_SUMMARY specific cytotoxic stress conditions, exogenous or released polyunsaturated PR:PROJECT_SUMMARY fatty acids (PUFAs) are channeled into overall depleting phospholipids that PR:PROJECT_SUMMARY become vulnerable to peroxidation in the presence of associated redox stress. In PR:PROJECT_SUMMARY fibroblasts, this reprogramming results from reduced growth factor receptor PR:PROJECT_SUMMARY tyrosine kinase (RTK) and phosphatidylinositol-3-kinase (PI3K)/Akt signaling, PR:PROJECT_SUMMARY which reduces de novo fatty acid biosynthesis by mechanisms that differ PR:PROJECT_SUMMARY depending on the specific cytotoxic stressors. We conclude that alterations in PR:PROJECT_SUMMARY PUFA metabolism during cytotoxic stress render cells prone to oxidative PR:PROJECT_SUMMARY modifications in phospholipids. PR:INSTITUTE University of Innsbruck PR:DEPARTMENT Michael Popp Institute PR:LAST_NAME Koeberle PR:FIRST_NAME Andreas PR:ADDRESS Mitterweg 24, Innsbruck, Tyrol, 6020, Austria PR:EMAIL andreas.koeberle@uibk.ac.at PR:PHONE +43 512 507 57903 PR:FUNDING_SOURCE Austrian Science Fund (FWF) (P 36299). German Research Council (GRK 1715 and KO PR:FUNDING_SOURCE 4589/4-1), the Phospholipid Research Center Heidelberg (AKO-2015-037/1-1, PR:FUNDING_SOURCE AKO-2019-070/2-1, AKO-2O22-100/2-2), the University of Jena (DRM/2013-05 and PR:FUNDING_SOURCE 2.7-05). PR:CONTRIBUTORS André Gollowitzer, Helmut Pein, Konstantin Loeser, Maria Thuermer, and Andreas PR:CONTRIBUTORS Koeberle #STUDY ST:STUDY_TITLE Analysis of oxidized arachidonoyl-phosphatidylethanolamine in ST:STUDY_TITLE valinomycin-treated fibroblasts ST:STUDY_SUMMARY Treatment of NIH-3T3 fibroblasts with valinomycin for 48 h and analysis of ST:STUDY_SUMMARY isomeric arachidonoyl phosphatidylethanolamines with two oxygens incorporated. ST:INSTITUTE University of Innsbruck ST:LAST_NAME Koeberle ST:FIRST_NAME Andreas ST:ADDRESS Mitterweg 24, Innsbruck, Tyrol, 6020, Austria ST:EMAIL andreas.koeberle@uibk.ac.at ST:PHONE +43 512 507 57903 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - 211109_DMSO_3h_DMSO_dil_1_5_n1_1 Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:- RAW_FILE_NAME(Local_data_file)=211109_PUFA_PC_LipidROS_oxPE_n1_n3_std_0.5µl; concentration A=-; concentration B=- SUBJECT_SAMPLE_FACTORS - 211109_DMSO_3h_DMSO_dil_1_5_n2_9 Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:- RAW_FILE_NAME(Local_data_file)=211109_PUFA_PC_LipidROS_oxPE_n1_n3_std_0.5µl; concentration A=-; concentration B=- SUBJECT_SAMPLE_FACTORS - 211109_VAL_10µM_3h_DMSO_dil_UD_n2_13 Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:VAL RAW_FILE_NAME(Local_data_file)=211109_PUFA_PC_LipidROS_oxPE_n1_n3_std_0.5µl; concentration A=-; concentration B=10 µM SUBJECT_SAMPLE_FACTORS - 211109_DMSO_3h_DMSO_dil_1_5_n3_17 Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:- RAW_FILE_NAME(Local_data_file)=211109_PUFA_PC_LipidROS_oxPE_n1_n3_std_0.5µl; concentration A=-; concentration B=- SUBJECT_SAMPLE_FACTORS - 211109_VAL_10µM_3h_DMSO_dil_UD_n3_21 Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:VAL RAW_FILE_NAME(Local_data_file)=211109_PUFA_PC_LipidROS_oxPE_n1_n3_std_0.5µl; concentration A=-; concentration B=10 µM SUBJECT_SAMPLE_FACTORS - 211124_DMSO_3h_DMSO_dil_1_5_n4_25 Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:- RAW_FILE_NAME(Local_data_file)=211124_PUFA_PC_LipidROS_oxPE_n4_std_0.5µl; concentration A=-; concentration B=- SUBJECT_SAMPLE_FACTORS - 211124_VAL_10µM_3h_DMSO_dil_UD_n4_29 Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:VAL RAW_FILE_NAME(Local_data_file)=211124_PUFA_PC_LipidROS_oxPE_n4_std_0.5µl; concentration A=-; concentration B=10 µM #COLLECTION CO:COLLECTION_SUMMARY Cultured cells were washed, trypsinized, counted and flash-frozen in liquid N2 CO:COLLECTION_SUMMARY and stored at -80°C. CO:SAMPLE_TYPE Fibroblasts CO:COLLECTION_METHOD Trypsinization of cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For the analysis of Isomeric arachidonoyl phosphatidylethanolamines with two TR:TREATMENT_SUMMARY oxygens, NIH-3T3 fibroblasts were treated with VAL (10 µM) for 48h. TR:TREATMENT_VEHICLE DMSO TR:CELL_MEDIA DMEM + 10 % FCS TR:CELL_ENVIR_COND 37 °C, 5 % CO2 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Phospholipids were extracted from cell pellets by successive addition of PBS pH SP:SAMPLEPREP_SUMMARY 7.4, methanol, chloroform, and saline to a final ratio of 14:34:35:17. SP:SAMPLEPREP_SUMMARY Evaporation of the organic layer yielded a lipid film that was dissolved in SP:SAMPLEPREP_SUMMARY methanol, diluted, and subjected to UPLC-MS/MS. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation of oxidized phospholipids was carried out on an CH:CHROMATOGRAPHY_SUMMARY Acquity BEH C8 column (1.7 μm, 2.1×100 mm, Waters, Milford, MA) using an CH:CHROMATOGRAPHY_SUMMARY ExionLC AD UHPLC system (Sciex). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Sciex ExionLC AD CH:COLUMN_NAME Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um) CH:SOLVENT_A 5% Water, 95% Acetonitrile; 2 mM ammonium acetate CH:SOLVENT_B 90% Water, 10% Acetonitrile; 2 mM ammonium acetate CH:FLOW_GRADIENT The gradient was ramped from 75 to 85% A over 5 min and further increased to CH:FLOW_GRADIENT 100% A within 2 min, followed by isocratic elution for another 2 min. CH:FLOW_RATE 0.75 ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6500+ MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Targeted MRM with pre-optimized settings and subsequent automated integration of MS:MS_COMMENTS selected signals using Analyst 1.6 and Analyst 1.7 (Sciex). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol / 10^6 cells MS_METABOLITE_DATA_START Samples 211109_DMSO_3h_DMSO_dil_1_5_n1_1 211109_DMSO_3h_DMSO_dil_1_5_n2_9 211109_VAL_10µM_3h_DMSO_dil_UD_n2_13 211109_DMSO_3h_DMSO_dil_1_5_n3_17 211109_VAL_10µM_3h_DMSO_dil_UD_n3_21 211124_DMSO_3h_DMSO_dil_1_5_n4_25 211124_VAL_10µM_3h_DMSO_dil_UD_n4_29 Factors Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:- Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:- Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:VAL Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:- Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:VAL Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:- Sample source:cultured cells | treatment A:vehicle (DMSO) | treatment B:VAL PE(16:0/20:4+2[O])T4_1I 2.26E-06 4.17E-06 4.66E-05 2.20E-06 9.46E-05 6.34E-06 4.39E-05 PE(18:0/20:4+1[O])T3_2I 9.98E-05 8.21E-05 8.16E-05 7.58E-05 1.58E-04 7.61E-05 2.15E-04 PE(18:0/20:4+2[O])T4_1I 1.36E-06 5.56E-07 1.21E-05 5.50E-07 3.05E-05 2.77E-06 4.23E-05 PE(18:0/20:4+2[O])T4_2I 1.81E-06 1.67E-06 3.36E-05 1.92E-06 3.78E-05 3.17E-06 4.61E-05 PE(18:0/20:4+2[O])T4_3I 4.53E-07 2.79E-07 6.89E-06 5.49E-07 1.59E-05 1.19E-06 3.03E-05 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PE(16:0/20:4+2[O])T4_1I PE(18:0/20:4+1[O])T3_2I PE(18:0/20:4+2[O])T4_1I PE(18:0/20:4+2[O])T4_2I PE(18:0/20:4+2[O])T4_3I METABOLITES_END #END