#METABOLOMICS WORKBENCH Brodesser_20230816_054636 DATATRACK_ID:4224 STUDY_ID:ST003114 ANALYSIS_ID:AN005104 PROJECT_ID:PR001935 VERSION 1 CREATED_ON March 6, 2024, 10:51 am #PROJECT PR:PROJECT_TITLE Lipid unsaturation promotes BAX and BAK pore activity during apoptosis PR:PROJECT_SUMMARY BAX and BAK are proapoptotic members of the BCL2 family that directly mediate PR:PROJECT_SUMMARY mitochondrial outer membrane permeabilization (MOMP), a central step in PR:PROJECT_SUMMARY apoptosis execution. However, the molecular architecture of the mitochondrial PR:PROJECT_SUMMARY apoptotic pore remains a key open question and especially little is known about PR:PROJECT_SUMMARY the contribution of lipids to MOMP. By performing a comparative lipidomics PR:PROJECT_SUMMARY analysis of the proximal membrane environment of BAK isolated in lipid PR:PROJECT_SUMMARY nanodiscs, we find a significant enrichment of unsaturated species nearby BAK PR:PROJECT_SUMMARY and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids PR:PROJECT_SUMMARY promote BAX pore activity in model membranes, isolated mitochondria and cellular PR:PROJECT_SUMMARY systems, which is further supported by molecular dynamics simulations. PR:PROJECT_SUMMARY Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis PR:PROJECT_SUMMARY sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA PR:PROJECT_SUMMARY release. The correlation of FADS2 levels with the sensitization to apoptosis of PR:PROJECT_SUMMARY different lung and kidney cancer cell lines by co-treatment with unsaturated PR:PROJECT_SUMMARY fatty acids supports the relevance of our findings. Altogether, our work PR:PROJECT_SUMMARY provides new insight on how local lipid environment affects BAX and BAK function PR:PROJECT_SUMMARY during apoptosis. PR:INSTITUTE University of Cologne PR:DEPARTMENT Institute for Genetics, Cluster of Excellence Cellular Stress Responses in PR:DEPARTMENT Aging-associated Diseases (CECAD) PR:LAST_NAME García-Sáez PR:FIRST_NAME Ana J. PR:ADDRESS Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany PR:EMAIL ana.garcia@uni-koeln.de PR:PHONE +49 221 478 84261 PR:CONTRIBUTORS Shashank Dadsena, Rodrigo Cuevas Arenas, Gonçalo Vieira, Susanne Brodesser, PR:CONTRIBUTORS Manuel N. Melo, Ana J. García-Sáez #STUDY ST:STUDY_TITLE Lipidomics analyses in model membranes, isolated mitochondria and cellular ST:STUDY_TITLE systems to study how the local lipid environment affects BAX and BAK function ST:STUDY_TITLE during apoptosis. ST:STUDY_SUMMARY To investigate how the local lipid environment affects BAX and BAK function ST:STUDY_SUMMARY during apoptosis, we performed quantitative analyses of different lipid classes ST:STUDY_SUMMARY (glycerophospholipids, fatty acids, ceramides and sphingomyelins) in cultured ST:STUDY_SUMMARY cells, isolated mitochondria and lipid nanodics formed by Styrene-Malic Acid ST:STUDY_SUMMARY (SMA) co-polymers. Ceramides, sphingomyelins, fatty acids and cardiolipins were ST:STUDY_SUMMARY analyzed by Liquid Chromatography coupled to Tandem Mass Spectrometry ST:STUDY_SUMMARY (LC-MS/MS). For glycerophospholipids (PC, PE, PI, PS, PG, PA) we applied direct ST:STUDY_SUMMARY infusion MS approaches (Shotgun Lipidomics). ST:INSTITUTE University of Cologne ST:DEPARTMENT Faculty of Medicine and University Hospital of Cologne, Cluster of Excellence ST:DEPARTMENT Cellular Stress Responses in Aging-associated Diseases (CECAD) ST:LABORATORY CECAD Lipidomics/Metabolomics Facility ST:LAST_NAME Brodesser ST:FIRST_NAME Susanne ST:ADDRESS Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany ST:EMAIL susanne.brodesser@uk-koeln.de ST:PHONE +49 221 478 84015 #SUBJECT SU:SUBJECT_TYPE Cultured cells 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 - S01_mitos_control.SMA_1 Sample source:mitochondrial SMALPs | Genotype:WT | Condition:control RAW_FILE_NAME=GPL_S01_mitos_control.SMA_1.mzML; RAW_FILE_NAME=CerSM_S01_mitos_control.SMA_1.mzML; RAW_FILE_NAME=CL_S01_mitos_control.SMA_1.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S02_mitos_control.SMA_2 Sample source:mitochondrial SMALPs | Genotype:WT | Condition:control RAW_FILE_NAME=GPL_S02_mitos_control.SMA_2.mzML; RAW_FILE_NAME=CerSM_S02_mitos_control.SMA_2.mzML; RAW_FILE_NAME=CL_S02_mitos_control.SMA_2.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S03_mitos_control.SMA_3 Sample source:mitochondrial SMALPs | Genotype:WT | Condition:control RAW_FILE_NAME=-; RAW_FILE_NAME=CerSM_S03_mitos_control.SMA_3.mzML; RAW_FILE_NAME=CL_S03_mitos_control.SMA_3.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S04_mitos_control.SMA_4 Sample source:mitochondrial SMALPs | Genotype:WT | Condition:control RAW_FILE_NAME=GPL_S04_mitos_control.SMA_4.mzML; RAW_FILE_NAME=CerSM_S04_mitos_control.SMA_4.mzML; RAW_FILE_NAME=CL_S04_mitos_control.SMA_4.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S05_mitos_apoptosis.SMA_1 Sample source:mitochondrial SMALPs | Genotype:WT | Condition:apoptotic RAW_FILE_NAME=GPL_S05_mitos_apoptosis.SMA_1.mzML; RAW_FILE_NAME=CerSM_S05_mitos_apoptosis.SMA_1.mzML; RAW_FILE_NAME=CL_S05_mitos_apoptosis.SMA_1.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S06_mitos_apoptosis.SMA_2 Sample source:mitochondrial SMALPs | Genotype:WT | Condition:apoptotic RAW_FILE_NAME=GPL_S06_mitos_apoptosis.SMA_2.mzML; RAW_FILE_NAME=CerSM_S06_mitos_apoptosis.SMA_2.mzML; RAW_FILE_NAME=CL_S06_mitos_apoptosis.SMA_2.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S07_mitos_apoptosis.SMA_3 Sample source:mitochondrial SMALPs | Genotype:WT | Condition:apoptotic RAW_FILE_NAME=GPL_S07_mitos_apoptosis.SMA_3.mzML; RAW_FILE_NAME=CerSM_S07_mitos_apoptosis.SMA_3.mzML; RAW_FILE_NAME=CL_S07_mitos_apoptosis.SMA_3.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S08_mitos_apoptosis.SMA_4 Sample source:mitochondrial SMALPs | Genotype:WT | Condition:apoptotic RAW_FILE_NAME=-; RAW_FILE_NAME=CerSM_S08_mitos_apoptosis.SMA_4.mzML; RAW_FILE_NAME=CL_S08_mitos_apoptosis.SMA_4.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S09_mitos_control_1 Sample source:total mitochondria | Genotype:WT | Condition:control RAW_FILE_NAME=GPL_S09_mitos_control_1.mzML; RAW_FILE_NAME=CerSM_S09_mitos_control_1.mzML; RAW_FILE_NAME=CL_S09_mitos_control_1.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S10_mitos_control_2 Sample source:total mitochondria | Genotype:WT | Condition:control RAW_FILE_NAME=-; RAW_FILE_NAME=CerSM_S10_mitos_control_2.mzML; RAW_FILE_NAME=CL_S10_mitos_control_2.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S11_mitos_control_3 Sample source:total mitochondria | Genotype:WT | Condition:control RAW_FILE_NAME=GPL_S11_mitos_control_3.mzML; RAW_FILE_NAME=CerSM_S11_mitos_control_3.mzML; RAW_FILE_NAME=CL_S11_mitos_control_3.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S12_mitos_control_4 Sample source:total mitochondria | Genotype:WT | Condition:control RAW_FILE_NAME=GPL_S12_mitos_control_4.mzML; RAW_FILE_NAME=CerSM_S12_mitos_control_4.mzML; RAW_FILE_NAME=CL_S12_mitos_control_4.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S13_mitos_apoptosis_1 Sample source:total mitochondria | Genotype:WT | Condition:apoptotic RAW_FILE_NAME=GPL_S13_mitos_apoptosis_1.mzML; RAW_FILE_NAME=CerSM_S13_mitos_apoptosis_1.mzML; RAW_FILE_NAME=CL_S13_mitos_apoptosis_1.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S14_mitos_apoptosis_2 Sample source:total mitochondria | Genotype:WT | Condition:apoptotic RAW_FILE_NAME=-; RAW_FILE_NAME=CerSM_S14_mitos_apoptosis_2.mzML; RAW_FILE_NAME=CL_S14_mitos_apoptosis_2.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S15_mitos_apoptosis_3 Sample source:total mitochondria | Genotype:WT | Condition:apoptotic RAW_FILE_NAME=GPL_S15_mitos_apoptosis_3.mzML; RAW_FILE_NAME=CerSM_S15_mitos_apoptosis_3.mzML; RAW_FILE_NAME=CL_S15_mitos_apoptosis_3.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S16_mitos_apoptosis_4 Sample source:total mitochondria | Genotype:WT | Condition:apoptotic RAW_FILE_NAME=GPL_S16_mitos_apoptosis_4.mzML; RAW_FILE_NAME=CerSM_S16_mitos_apoptosis_4.mzML; RAW_FILE_NAME=CL_S16_mitos_apoptosis_4.mzML; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S02_pulldown_Control_22.08 Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:control RAW_FILE_NAME=GPL_S02_pulldown_Control_22.08.mzML; RAW_FILE_NAME=CerSM_S02_pulldown_Control_22.08.mzML; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S03_pulldown_Control_01.09 Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:control RAW_FILE_NAME=GPL_S03_pulldown_Control_01.09.mzML; RAW_FILE_NAME=CerSM_S03_pulldown_Control_01.09.mzML; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S04_pulldown_Control_04.09 Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:control RAW_FILE_NAME=GPL_S04_pulldown_Control_04.09.mzML; RAW_FILE_NAME=CerSM_S04_pulldown_Control_04.09.mzML; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S05_pulldown_Control_31.09 Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:control RAW_FILE_NAME=GPL_S05_pulldown_Control_31.09.mzML; RAW_FILE_NAME=CerSM_S05_pulldown_Control_31.09.mzML; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S06_pulldown_Apoptosis_22.08 Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:apoptotic RAW_FILE_NAME=GPL_S06_pulldown_Apoptosis_22.08.mzML; RAW_FILE_NAME=CerSM_S06_pulldown_Apoptosis_22.08.mzML; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S07_pulldown_Apoptosis_01.09 Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:apoptotic RAW_FILE_NAME=GPL_S07_pulldown_Apoptosis_01.09.mzML; RAW_FILE_NAME=CerSM_S07_pulldown_Apoptosis_01.09.mzML; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S08_pulldown_Apoptosis_04.09 Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:apoptotic RAW_FILE_NAME=GPL_S08_pulldown_Apoptosis_04.09.mzML; RAW_FILE_NAME=CerSM_S08_pulldown_Apoptosis_04.09.mzML; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S09_pulldown_Apoptosis_31.09 Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:apoptotic RAW_FILE_NAME=GPL_S09_pulldown_Apoptosis_31.09.mzML; RAW_FILE_NAME=CerSM_S09_pulldown_Apoptosis_31.09.mzML; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - S01_mitos_no.treatment_WT_1 Sample source:total mitochondria | Genotype:WT | Condition:untreated RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S01_mitos_no.treatment_WT_1.mzML SUBJECT_SAMPLE_FACTORS - S02_mitos_no.treatment_WT_2 Sample source:total mitochondria | Genotype:WT | Condition:untreated RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S02_mitos_no.treatment_WT_2.mzML SUBJECT_SAMPLE_FACTORS - S03_mitos_no.treatment_WT_3 Sample source:total mitochondria | Genotype:WT | Condition:untreated RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S03_mitos_no.treatment_WT_3.mzML SUBJECT_SAMPLE_FACTORS - S04_mitos_no.treatment_WT_4 Sample source:total mitochondria | Genotype:WT | Condition:untreated RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S04_mitos_no.treatment_WT_4.mzML SUBJECT_SAMPLE_FACTORS - S05_mitos_linoleic.acid_WT_1 Sample source:total mitochondria | Genotype:WT | Condition:linoleic acid RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S05_mitos_linoleic.acid_WT_1.mzML SUBJECT_SAMPLE_FACTORS - S06_mitos_linoleic.acid_WT_2 Sample source:total mitochondria | Genotype:WT | Condition:linoleic acid RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S06_mitos_linoleic.acid_WT_2.mzML SUBJECT_SAMPLE_FACTORS - S07_mitos_linoleic.acid_WT_3 Sample source:total mitochondria | Genotype:WT | Condition:linoleic acid RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S07_mitos_linoleic.acid_WT_3.mzML SUBJECT_SAMPLE_FACTORS - S08_mitos_linoleic.acid_WT_4 Sample source:total mitochondria | Genotype:WT | Condition:linoleic acid RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S08_mitos_linoleic.acid_WT_4.mzML SUBJECT_SAMPLE_FACTORS - S09_smitos_no.treatment_KO_1 Sample source:total mitochondria | Genotype:FADS2 KO | Condition:untreated RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S09_mitos_no.treatment_KO_1.mzML SUBJECT_SAMPLE_FACTORS - S10_mitos_no.treatment_KO_2 Sample source:total mitochondria | Genotype:FADS2 KO | Condition:untreated RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S10_mitos_no.treatment_KO_2.mzML SUBJECT_SAMPLE_FACTORS - S11_mitos_no.treatment_KO_3 Sample source:total mitochondria | Genotype:FADS2 KO | Condition:untreated RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S11_mitos_no.treatment_KO_3.mzML SUBJECT_SAMPLE_FACTORS - S12_mitos_no.treatment_KO_4 Sample source:total mitochondria | Genotype:FADS2 KO | Condition:untreated RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S12_mitos_no.treatment_KO_4.mzML SUBJECT_SAMPLE_FACTORS - S13_mitos_linoleic.acid_KO_1 Sample source:total mitochondria | Genotype:FADS2 KO | Condition:linoleic acid RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S13_mitos_linoleic.acid_KO_1.mzML SUBJECT_SAMPLE_FACTORS - S14_mitos_linoleic.acid_KO_2 Sample source:total mitochondria | Genotype:FADS2 KO | Condition:linoleic acid RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S14_mitos_linoleic.acid_KO_2.mzML SUBJECT_SAMPLE_FACTORS - S15_mitos_linoleic.acid_KO_3 Sample source:total mitochondria | Genotype:FADS2 KO | Condition:linoleic acid RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S15_mitos_linoleic.acid_KO_3.mzML SUBJECT_SAMPLE_FACTORS - S16_mitos_linoleic.acid_KO_4 Sample source:total mitochondria | Genotype:FADS2 KO | Condition:linoleic acid RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=-; RAW_FILE_NAME=FA_S16_mitos_linoleic.acid_KO_4.mzML #COLLECTION CO:COLLECTION_SUMMARY Human osteosarcoma U2OS WT, U2OS BAK Ko expressing GFP BAK, and U2OS FADS2 KO CO:COLLECTION_SUMMARY cell lines were cultured at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS CO:COLLECTION_SUMMARY and 1% penicillin/streptomycin (Invitrogen, Germany). For lipidomic experiments CO:COLLECTION_SUMMARY cells were incubated with 1 μM of ABT-737 and S63845 in the complete media and CO:COLLECTION_SUMMARY incubated for 50 min at 37°C and 5% CO2. FADS2 KO in U2OS cells was generated CO:COLLECTION_SUMMARY in the lab by the CRISPR/Cas9 method. Linoleic acid stock (50 mM) was prepared CO:COLLECTION_SUMMARY in ethanol and diluted into culture media before adding them to the cells. CO:COLLECTION_SUMMARY Mitochondria were isolated from cultured human osteosarcoma cells by mechanical CO:COLLECTION_SUMMARY disruption of cells followed by differential centrifugation: Cells were CO:COLLECTION_SUMMARY harvested by trypsinization, washed in PBS, and then resuspend in isolation CO:COLLECTION_SUMMARY buffer (IM;250 mM sucrose, 5 mM Tris, and 2 mM EDTA; pH 7.4 and protease CO:COLLECTION_SUMMARY inhibitor cocktail) and mechanically broken using glass homogenizer on ice CO:COLLECTION_SUMMARY (30-40 strokes on ice) and total cellular lysates were spin down first to remove CO:COLLECTION_SUMMARY nuclei and cell debris at 600 x g for 5 min and later at 10,800 x g for 10 min CO:COLLECTION_SUMMARY at 4°C to get the crude mitochondria. Mitochondrial pellet was washed 2-3 times CO:COLLECTION_SUMMARY with isolation buffer to remove other impurities from mitochondria. Isolated CO:COLLECTION_SUMMARY mitochondria were solubilized using SMA co-polymer. For this, mitochondria CO:COLLECTION_SUMMARY either from apoptotic or healthy cells were incubated with 0.5% SMA (2:1) for 45 CO:COLLECTION_SUMMARY min at room temperature with gentle rotation. Mitochondrial membrane was spun CO:COLLECTION_SUMMARY down at 100,000 x g for 40 min to separate solubilized SMALP from the CO:COLLECTION_SUMMARY insolubilized membrane. Next, the size of SMALP was analyzed by Dynamic Light CO:COLLECTION_SUMMARY Scattering (DLS). For DLS measurements, 15 μl of sample was added to a quartz CO:COLLECTION_SUMMARY cuvette which had been thoroughly cleaned with Milli-Q H2O. The cuvette was CO:COLLECTION_SUMMARY placed in DynaPro NanoStar (Wyatt Technology corporation, USA) and the sample CO:COLLECTION_SUMMARY was analyzed using 10 runs with 10 second acquisition time. This helps to CO:COLLECTION_SUMMARY determine the mass distribution of the sample as well as the estimated size of CO:COLLECTION_SUMMARY the particles. The distance distribution is shown on a log scale. The size of CO:COLLECTION_SUMMARY SMALP as well as the homogeneity with in the sample were also checked by CO:COLLECTION_SUMMARY Negative Transmission Electron Microscopy (TEM). For this the diluted SMALPs CO:COLLECTION_SUMMARY were placed onto a glow-discharged copper grid (Electron Microscopy Sciences) CO:COLLECTION_SUMMARY coated with a layer of thin carbon, washed twice with water, stained with 2% CO:COLLECTION_SUMMARY uranyl acetate for 5 min and then air-dried. The grids were imaged on a JEOL CO:COLLECTION_SUMMARY JEM2100PLUS electron microscope and recorded with a GATAN OneView camera (CECAD CO:COLLECTION_SUMMARY Imaging Facility). mEGFP-BAK-SMALPs were affinity purified from total CO:COLLECTION_SUMMARY solubilized mitochondrial membrane fraction (SMALP). For this total SMALP were CO:COLLECTION_SUMMARY incubated with 25 μl of GFP-trap MA beads for 90 min with slow rotation in cold CO:COLLECTION_SUMMARY room. Beads were washed 2 times with 100 μl of Tris buffer (50 mM Tris 150 mM CO:COLLECTION_SUMMARY NaCl pH 8), and finally resuspend in 100 ul of Tris buffer. Small aliquots of CO:COLLECTION_SUMMARY unbound and wash fractions were used to analyze the purification quality. CO:SAMPLE_TYPE Mitochondria #TREATMENT TR:TREATMENT_SUMMARY The samples were not subjected to any further treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Glycerophospholipids: Lipids from isolated mitochondria treated with or without SP:SAMPLEPREP_SUMMARY SMA were extracted using a procedure previously described (Ejsing et al., 2009) SP:SAMPLEPREP_SUMMARY with some modifications: 30-100 µl of sample were brought to a volume of 200 SP:SAMPLEPREP_SUMMARY µl with 155 mM ammonium carbonate buffer. Lipids were extracted by adding 990 SP:SAMPLEPREP_SUMMARY µl of chloroform/methanol 17:1 (v/v) and internal standards (125 pmol PC SP:SAMPLEPREP_SUMMARY 17:0-20:4, 138 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 118 pmol PS 17:0-20:4, SP:SAMPLEPREP_SUMMARY 61 pmol PG 17:0/20:4, 72 pmol PA 17:0/20:4, 10 µl Cardiolipin Mix I; Avanti SP:SAMPLEPREP_SUMMARY Polar Lipids), followed by shaking at 900 rpm/min in a ThermoMixer (Eppendorf) SP:SAMPLEPREP_SUMMARY at 20 °C for 30 min. After centrifugation (12,000xg, 5 min, 4 °C), the lower SP:SAMPLEPREP_SUMMARY (organic) phase was transferred to a new tube, and the upper phase was extracted SP:SAMPLEPREP_SUMMARY again with 990 mL chloroform/methanol 2:1 (v/v). The combined organic phases SP:SAMPLEPREP_SUMMARY were dried under a stream of nitrogen. The residues were resolved in 200 µl of SP:SAMPLEPREP_SUMMARY methanol. Ceramides and sphingomyelins: For the analysis of ceramides and SP:SAMPLEPREP_SUMMARY sphingomyelins in isolated mitochondria without and after SMA treatment, lipids SP:SAMPLEPREP_SUMMARY were extracted as described above in the presence of 127 pmol ceramide 12:0 and SP:SAMPLEPREP_SUMMARY 124 pmol sphingomyelin 12:0 (internal standards, Avanti Polar Lipids). The dried SP:SAMPLEPREP_SUMMARY extracts were resolved in 100 µL of Milli-Q water and 750 µL of SP:SAMPLEPREP_SUMMARY chloroform/methanol 1:2 (v/v). Alkaline hydrolysis of glycerolipids was SP:SAMPLEPREP_SUMMARY conducted as previously published (Schwamb et al., 2012; Oteng et al., 2017). SP:SAMPLEPREP_SUMMARY Fatty acids: To 100 µl of a suspension of isolated mitochondria in PBS, 500 µl SP:SAMPLEPREP_SUMMARY of methanol, 250 µl of chloroform, and 0.5 µg palmitic-d31 acid SP:SAMPLEPREP_SUMMARY (Sigma-Aldrich) as internal standard were added. The mixture was sonicated for 5 SP:SAMPLEPREP_SUMMARY min, and lipids were extracted in a shaking bath at 48 °C for 1 h. SP:SAMPLEPREP_SUMMARY Glycerolipids were degraded by alkaline hydrolysis adding 75 µl of 1 M SP:SAMPLEPREP_SUMMARY potassium hydroxide in methanol. After 5 min of sonication, the extract was SP:SAMPLEPREP_SUMMARY incubated for 1.5 h at 37 °C, and then neutralized with 6 µl of glacial acetic SP:SAMPLEPREP_SUMMARY acid. 2 ml of chloroform and 4 ml of water were added to the extract which was SP:SAMPLEPREP_SUMMARY vortexed vigorously for 30 sec and then centrifuged (4,000 × g, 5 min, 4 °C) SP:SAMPLEPREP_SUMMARY to separate layers. The lower (organic) phase was transferred to a new tube, and SP:SAMPLEPREP_SUMMARY the upper phase extracted with additional 2 ml of chloroform. The combined SP:SAMPLEPREP_SUMMARY organic phases were dried under a stream of nitrogen. The residues were resolved SP:SAMPLEPREP_SUMMARY in 200 µl of acetonitrile/water 2:1 (v/v) and sonicated for 5 min. After SP:SAMPLEPREP_SUMMARY centrifugation (12,000 × g, 20 min, 4 °C), 40 µl of the clear supernatants SP:SAMPLEPREP_SUMMARY were transferred to autoinjector vials. References: Ejsing et al., Proc Natl SP:SAMPLEPREP_SUMMARY Acad Sci USA 2009, 106, 2136; Oteng et al., J Lipid Res 2017, 58, 1100; Schwamb SP:SAMPLEPREP_SUMMARY et al., Blood 2012, 120, 3978. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Waters Acquity BEH Shield RP18 (100×2.1 mm, 1.7 μm) CH:SOLVENT_A 60% acetonitrile/40% water; 10 mM ammonium formate CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 10 mM ammonium formate CH:FLOW_GRADIENT 0 min: 30% B, 0.5 min: 30% B, 4.5 min: 68% B, 20.5 min: 75% B, 21 min: 97% B, 24 CH:FLOW_GRADIENT min: 97% B, 24.5 min: 30% B, 28 min: 30% B CH:FLOW_RATE 0.4 ml/min CH:COLUMN_TEMPERATURE 50 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME SCIEX QTRAP 6500 MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Cardiolipin (CL) species were analyzed by Liquid Chromatography coupled to MS:MS_COMMENTS Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). CL species were MS:MS_COMMENTS monitored in the positive ion mode using the following Multiple Reaction MS:MS_COMMENTS Monitoring (MRM) transitions: CL 68:4, m/z 1418.9 to 575.4; CL 70:4, m/z 1446.9 MS:MS_COMMENTS to 575.4; CL 72:4 m/z 1475.0 to 603.4; CL 61:1 (internal standard), m/z 1326.9 MS:MS_COMMENTS to 535.4. For all MRM transitions the values for declustering potential, MS:MS_COMMENTS entrance potential, collision energy, and cell exit potential were 140 V, 10 V, MS:MS_COMMENTS 45 V, and 7 V, respectively (Tatsuta, Methods Mol Biol 2017, 1567, 79). The MS:MS_COMMENTS instrument settings for nebulizer gas (Gas 1), turbo gas (Gas 2), curtain gas, MS:MS_COMMENTS and collision gas were 50 psi, 50 psi, 40 psi, and medium, respectively. The MS:MS_COMMENTS Turbo V ESI source temperature was 500 °C, and the ionspray voltage was 4.5 kV. MS:MS_COMMENTS The LC chromatogram peaks of the endogenous CL species and the internal standard MS:MS_COMMENTS CL 61:1 were integrated using the MultiQuant 3.0.2 software (SCIEX). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS counts per second (cps) MS_METABOLITE_DATA_START Samples S01_mitos_control.SMA_1 S02_mitos_control.SMA_2 S03_mitos_control.SMA_3 S04_mitos_control.SMA_4 S05_mitos_apoptosis.SMA_1 S06_mitos_apoptosis.SMA_2 S07_mitos_apoptosis.SMA_3 S08_mitos_apoptosis.SMA_4 S09_mitos_control_1 S10_mitos_control_2 S11_mitos_control_3 S12_mitos_control_4 S13_mitos_apoptosis_1 S14_mitos_apoptosis_2 S15_mitos_apoptosis_3 S16_mitos_apoptosis_4 Factors Sample source:mitochondrial SMALPs | Genotype:WT | Condition:control Sample source:mitochondrial SMALPs | Genotype:WT | Condition:control Sample source:mitochondrial SMALPs | Genotype:WT | Condition:control Sample source:mitochondrial SMALPs | Genotype:WT | Condition:control Sample source:mitochondrial SMALPs | Genotype:WT | Condition:apoptotic Sample source:mitochondrial SMALPs | Genotype:WT | Condition:apoptotic Sample source:mitochondrial SMALPs | Genotype:WT | Condition:apoptotic Sample source:mitochondrial SMALPs | Genotype:WT | Condition:apoptotic Sample source:total mitochondria | Genotype:WT | Condition:control Sample source:total mitochondria | Genotype:WT | Condition:control Sample source:total mitochondria | Genotype:WT | Condition:control Sample source:total mitochondria | Genotype:WT | Condition:control Sample source:total mitochondria | Genotype:WT | Condition:apoptotic Sample source:total mitochondria | Genotype:WT | Condition:apoptotic Sample source:total mitochondria | Genotype:WT | Condition:apoptotic Sample source:total mitochondria | Genotype:WT | Condition:apoptotic CL 61:1 (IS) 1213956 1395870 1377231 1106401 1255664 1328738 1326949 1305189 1373051 1590241 1840183 1729046 1364377 1294713 1218141 1079713 CL 68:4 19852 27420 30494 21763 37263 25501 40739 50478 153002 35435 308215 112051 102760 153878 131520 244514 CL 70:4 7481 12763 10683 9879 11573 12156 17787 17077 74824 15356 139558 47175 47814 67604 79936 126598 CL 72:4 6156 7129 5710 5040 7800 8516 8967 13180 36852 9586 72140 22803 21082 32437 36884 73689 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Q1 Mass (Da) Q3 Mass (Da) RT (min) CE (volts) KEGG_ID LM_ID CL 61:1 (IS) 1326.9 535.4 7.84 45 C05980 LMGP12010000 CL 68:4 1418.9 575.4 8.12 45 C05980 LMGP12010000 CL 70:4 1446.9 575.4 8.78 45 C05980 LMGP12010000 CL 72:4 1475 603.4 9.46 45 C05980 LMGP12010000 METABOLITES_END #END