#METABOLOMICS WORKBENCH Andre_Gollowitzer_20240705_020100 DATATRACK_ID:4978 STUDY_ID:ST003319 ANALYSIS_ID:AN005433 PROJECT_ID:PR002065 VERSION 1 CREATED_ON July 14, 2024, 12:02 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 Changes in the phosphatidylcholine fatty acid composition in subcellular ST:STUDY_TITLE fractions of fibroblasts induced by valinomycin ST:STUDY_SUMMARY Determination of the impact of valinomycin on the subcellular ST:STUDY_SUMMARY phosphatidylcholine fatty acid composition of NIH-3T3 fibroblasts challenged ST:STUDY_SUMMARY with valinomycin for 48 h and disrupted by repeated passage through a syringe. ST:STUDY_SUMMARY Nuclear, mitochondrial, membrane, and cytosolic fractions were obtained by ST:STUDY_SUMMARY differential centrifugation. ST:INSTITUTE University of Innsbruck ST:DEPARTMENT Michael Popp Institute 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 - 180208_nucleus_DMSO Sample source:cultured cells | treatment:vehicle RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_nuclei; concentration=- SUBJECT_SAMPLE_FACTORS - 180208_nucleus_Valinomycin Sample source:cultured cells | treatment:VAL RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_nuclei; concentration=10 µM SUBJECT_SAMPLE_FACTORS - 180222_nucleus_DMSO Sample source:cultured cells | treatment:vehicle RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_nuclei; concentration=- SUBJECT_SAMPLE_FACTORS - 180222_nucleus_Valinomycin Sample source:cultured cells | treatment:VAL RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_nuclei; concentration=10 µM SUBJECT_SAMPLE_FACTORS - 180405_nucleus_DMSO Sample source:cultured cells | treatment:vehicle RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_nuclei; concentration=- SUBJECT_SAMPLE_FACTORS - 180205_nucleus_Valinomycin Sample source:cultured cells | treatment:VAL RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_nuclei; concentration=10 µM SUBJECT_SAMPLE_FACTORS - 180208_membrane_DMSO Sample source:cultured cells | treatment:vehicle RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_membrane; concentration=- SUBJECT_SAMPLE_FACTORS - 180208_membrane_Valinomycin Sample source:cultured cells | treatment:VAL RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_membrane; concentration=10 µM SUBJECT_SAMPLE_FACTORS - 180222_membrane_DMSO Sample source:cultured cells | treatment:vehicle RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_membrane; concentration=- SUBJECT_SAMPLE_FACTORS - 180222_membrane_Valinomycin Sample source:cultured cells | treatment:VAL RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_membrane; concentration=10 µM SUBJECT_SAMPLE_FACTORS - 180405_membrane_DMSO Sample source:cultured cells | treatment:vehicle RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_membrane; concentration=- SUBJECT_SAMPLE_FACTORS - 180205_membrane_Valinomycin Sample source:cultured cells | treatment:VAL RAW_FILE_NAME(Local_data_file)=180724_apoptosis_SCF_membrane; concentration=10 µM #COLLECTION CO:COLLECTION_SUMMARY To separate (peri)nuclear from non-nuclear membranes, NIH-3T3 fibroblasts were CO:COLLECTION_SUMMARY suspended in 500 µl ice-cold hypotonic fractionation buffer (10 mM HEPES pH CO:COLLECTION_SUMMARY 7.4, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM KCl, 1 mM dithiothreitol) and CO:COLLECTION_SUMMARY passed through a 25-gauge needle ten times. After 45 min on ice, the CO:COLLECTION_SUMMARY (peri)nuclear (pellet) and non-nuclear fractions (supernatant) were separated by CO:COLLECTION_SUMMARY centrifugation (600×g, 10 min, 4°C). Non-nuclear membranes were obtained from CO:COLLECTION_SUMMARY the supernatant by centrifugation (100.000×g, 1 h, 4°C). The remaining intact CO:COLLECTION_SUMMARY cells in the (peri)nuclear fraction were disrupted by repeated homogenization in CO:COLLECTION_SUMMARY fractionation buffer (500 µl). Pellets were washed with PBS pH 7.4 prior to CO:COLLECTION_SUMMARY analysis by UPLC-MS/MS and Western Blot. CO:SAMPLE_TYPE Fibroblasts CO:COLLECTION_METHOD Trypsinization of cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY NIH-3T3 fibroblasts were cultivated in DMEM high glucose medium containing TR:TREATMENT_SUMMARY heat-inactivated fetal calf serum (FCS, 10%). After cultivation for 24 h, cells TR:TREATMENT_SUMMARY were treated with vehicle or VAL (10 µM). 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 after subcellular fractionation SP:SAMPLEPREP_SUMMARY by successive addition of PBS pH 7.4, methanol, chloroform, and saline to a SP:SAMPLEPREP_SUMMARY final ratio of 14:34:35:17. Evaporation of the organic layer yielded a lipid SP:SAMPLEPREP_SUMMARY film that was dissolved in methanol, diluted, and subjected to UPLC-MS/MS. SP:EXTRACT_STORAGE -20℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation of phospholipids was carried out on an Acquity BEH C8 CH:CHROMATOGRAPHY_SUMMARY column (1.7 μm, 2.1×100 mm, Waters, Milford, MA) using an Acquity CH:CHROMATOGRAPHY_SUMMARY Ultraperformance LC system (Waters). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity H-Class CH:COLUMN_NAME Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um) CH:SOLVENT_A 90% Water/10% Acetonitrile; 10 mM ammonium acetate CH:SOLVENT_B 5% Water/95% Acetonitrile; 10 mM ammonium acetate CH:FLOW_GRADIENT The gradient was ramped from 70 to 80% B over 5 min and further increased to CH:FLOW_GRADIENT 100% B 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 5500 QTrap 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 (Sciex). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS relative units MS_METABOLITE_DATA_START Samples 180208_nucleus_DMSO 180222_nucleus_DMSO 180405_nucleus_DMSO 180208_nucleus_Valinomycin 180222_nucleus_Valinomycin 180205_nucleus_Valinomycin 180208_membrane_DMSO 180222_membrane_DMSO 180405_membrane_DMSO 180208_membrane_Valinomycin 180222_membrane_Valinomycin 180205_membrane_Valinomycin Factors Sample source:cultured cells | treatment:vehicle Sample source:cultured cells | treatment:vehicle Sample source:cultured cells | treatment:vehicle Sample source:cultured cells | treatment:VAL Sample source:cultured cells | treatment:VAL Sample source:cultured cells | treatment:VAL Sample source:cultured cells | treatment:vehicle Sample source:cultured cells | treatment:vehicle Sample source:cultured cells | treatment:vehicle Sample source:cultured cells | treatment:VAL Sample source:cultured cells | treatment:VAL Sample source:cultured cells | treatment:VAL PC(14:0_16:0) 2.545146877 2.404912559 2.388990143 8.486332109 6.104423456 10.2734089 3.133331603 3.12860383 3.127101021 7.301395706 6.484086845 8.590158993 PC(16:0/16:0) 13.06967315 12.60827944 11.03279084 14.19828641 15.03596928 12.48701634 17.6134025 20.55382142 17.25768876 17.34609038 15.53164988 17.33102253 PC(16:0_16:1) 3.557309534 3.303836186 3.04053291 3.598531212 2.809115219 3.292031581 3.5226805 2.692810781 2.97074597 3.270856458 3.006806783 2.727752242 PC(16:0_18:1) 18.4744252 18.56218917 16.07138824 19.17584659 24.30965093 17.65210037 18.37355987 20.16355899 17.02315618 19.81505945 23.55382244 20.04370432 PC(16:1_18:1) 9.355134465 9.070956595 11.11966321 6.217870257 5.510187544 7.832764796 8.454433201 7.089767514 8.970871054 6.119666921 5.880915975 6.570718107 PC(16:1_18:1) iso 3.311638986 3.011977865 2.92759883 2.015503876 1.818722033 1.912783867 2.985008213 2.23750461 2.697124631 2.152434572 1.927130927 1.853665888 PC(18:0_18:1) 12.2835274 13.77571272 16.85323956 15.58547532 13.9375332 16.74395373 11.5877648 12.87866026 15.71368263 16.18546389 16.01418658 15.22115892 PC(18:1/18:1) 29.18566109 30.00303533 28.92849883 12.97429621 14.96394068 14.58710545 27.43982705 26.66793285 26.77580249 14.47617761 14.41578378 15.37186346 PC(18:1_18:2) 3.095448904 2.82518854 3.205590411 2.643818849 2.268900754 3.08202267 2.428795503 1.606580345 2.091248808 2.257946071 2.270938323 2.456484063 PC(16:0_20:3 n-6) 0.39896897 0.347895118 0.445655252 0.987352101 0.972386037 0.868415227 0.302208906 0.24651577 0.300983473 0.694265661 0.699678208 0.625423857 PC(16:0_20:3 n-9) 0.259428099 0.221812323 0.166794948 0.263565891 0.280911522 0.204900586 0.226193169 0.191228592 0.129579249 0.221574147 0.237046152 0.182352498 PC(18:0_20:3 n-6) 0.122835274 0.098181139 0.140733238 0.624235006 0.515004457 0.497777879 0.087139991 0.059710152 0.099090014 0.352408405 0.358886667 0.304423178 PC(18:0_20:3 n-9) 0.231912997 0.185621892 0.204150067 0.252957976 0.217886501 0.25087551 0.202090618 0.191228592 0.178244758 0.209756859 0.231316028 0.210986361 PC(18:1_20:3 n-6) 0.326250488 0.248663289 0.28494137 0.562219502 0.502399453 0.483020496 0.292938694 0.175618094 0.228669262 0.379841395 0.428251317 0.406902268 PC(18:1_20:3 n-9) 0.339025356 0.258002755 0.200675172 0.198286414 0.175569701 0.184467287 0.318895287 0.275135015 0.194271151 0.204270261 0.176427479 0.18838068 PC(16:0_20:4) 0.728167504 0.733148101 0.769689188 3.541411669 2.881143814 2.866337842 0.760157371 0.4279878 0.517926107 2.658889766 2.786649416 2.426343154 PC(18:0_20:4) 0.325267805 0.304700086 0.316215423 2.252141983 2.070822116 1.356544048 0.331873584 0.2887942 0.293165721 1.686073748 1.676815017 1.186044759 PC(18:1_20:4) 1.061296767 0.833547363 0.744496201 2.888616891 2.647050879 2.304422107 0.841735235 0.425386051 0.586331441 2.215741472 2.102050479 1.959159069 PC(16:0_22:5) 0.306596844 0.30936982 0.335327344 1.109751122 0.912962446 0.953553975 0.283668482 0.204237339 0.244304767 0.79766693 0.73586846 0.785170673 PC(18:0_22:5) 0.132662096 0.105769455 0.15550154 0.599755202 0.614043775 0.459181646 0.100674501 0.119680479 0.10456244 0.546549563 0.431267172 0.355662723 PC(18:1_22:5) 0.348852178 0.29185832 0.248454975 0.526315789 0.437573717 0.491534371 0.209506788 0.109273481 0.166713573 0.278550356 0.276855429 0.287845679 PC(16:0_22:6) 0.225034222 0.197296225 0.179825804 0.609547124 0.446577291 0.601647151 0.194674449 0.11187523 0.113552856 0.352408405 0.307617143 0.399367041 PC(18:0_22:6) 0.086770838 0.08207056 0.075057727 0.345981232 0.342135828 0.252578285 0.070268206 0.029855076 0.088926935 0.234235527 0.227093832 0.197422952 PC(18:1_22:6) 0.228964951 0.215975157 0.164188777 0.341901265 0.22508936 0.361555882 0.239171466 0.124233541 0.126256704 0.242676447 0.238855664 0.317986587 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Exact mass Formula Super class Main class Sub class PC(14:0_16:0) PC 14:0_16:0 705.5309 C38H76NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:0/16:0) PC 16:0/16:0 733.5622 C40H80NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:0_16:1) PC 16:0_16:1 731.5465 C40H78NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:0_18:1) PC 16:0_18:1 759.5778 C42H82NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:1_18:1) PC 16:1_18:1 757.5622 C42H80NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:1_18:1) iso PC 16:1_18:1 757.5622 C42H80NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:0_18:1) PC 18:0_18:1 787.6091 C44H86NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:1/18:1) PC 18:1/18:1 785.5935 C44H84NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:1_18:2) PC 18:1_18:2 783.5778 C44H82NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:0_20:3 n-6) PC 16:0_20:3 783.5778 C44H82NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:0_20:3 n-9) PC 16:0_20:3 783.5778 C44H82NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:0_20:3 n-6) PC 18:0_20:3 811.6091 C46H86NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:0_20:3 n-9) PC 18:0_20:3 811.6091 C46H86NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:1_20:3 n-6) PC 18:1_20:3 809.5935 C46H84NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:1_20:3 n-9) PC 18:1_20:3 809.5935 C46H84NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:0_20:4) PC 16:0_20:4 781.5622 C44H80NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:0_20:4) PC 18:0_20:4 809.5935 C46H84NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:1_20:4) PC 18:1_20:4 807.5778 C46H82NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:0_22:5) PC 16:0_22:5 807.5778 C46H82NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:0_22:5) PC 18:0_22:5 835.6091 C48H86NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:1_22:5) PC 18:1_22:5 833.5935 C48H84NO8P Glycerophospholipids Glycerophosphocholines PC PC(16:0_22:6) PC 16:0_22:6 805.5622 C46H80NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:0_22:6) PC 18:0_22:6 833.5935 C48H84NO8P Glycerophospholipids Glycerophosphocholines PC PC(18:1_22:6) PC 18:1_22:6 831.5778 C48H82NO8P Glycerophospholipids Glycerophosphocholines PC METABOLITES_END #END