{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003114","ANALYSIS_ID":"AN005105","VERSION":"1","CREATED_ON":"March 6, 2024, 10:51 am"},

"PROJECT":{"PROJECT_TITLE":"Lipid unsaturation promotes BAX and BAK pore activity during apoptosis","PROJECT_SUMMARY":"BAX and BAK are proapoptotic members of the BCL2 family that directly mediate mitochondrial outer membrane permeabilization (MOMP), a central step in apoptosis execution. However, the molecular architecture of the mitochondrial apoptotic pore remains a key open question and especially little is known about the contribution of lipids to MOMP. By performing a comparative lipidomics analysis of the proximal membrane environment of BAK isolated in lipid nanodiscs, we find a significant enrichment of unsaturated species nearby BAK and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids promote BAX pore activity in model membranes, isolated mitochondria and cellular systems, which is further supported by molecular dynamics simulations. Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA release. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides new insight on how local lipid environment affects BAX and BAK function during apoptosis.","INSTITUTE":"University of Cologne","DEPARTMENT":"Institute for Genetics, Cluster of Excellence Cellular Stress Responses in Aging-associated Diseases (CECAD)","LAST_NAME":"García-Sáez","FIRST_NAME":"Ana J.","ADDRESS":"Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany","EMAIL":"ana.garcia@uni-koeln.de","PHONE":"+49 221 478 84261","CONTRIBUTORS":"Shashank Dadsena, Rodrigo Cuevas Arenas, Gonçalo Vieira, Susanne Brodesser, Manuel N. Melo, Ana J. García-Sáez"},

"STUDY":{"STUDY_TITLE":"Lipidomics analyses in model membranes, isolated mitochondria and cellular systems to study how the local lipid environment affects BAX and BAK function during apoptosis.","STUDY_SUMMARY":"To investigate how the local lipid environment affects BAX and BAK function during apoptosis, we performed quantitative analyses of different lipid classes (glycerophospholipids, fatty acids, ceramides and sphingomyelins) in cultured cells, isolated mitochondria and lipid nanodics formed by Styrene-Malic Acid (SMA) co-polymers. Ceramides, sphingomyelins, fatty acids and cardiolipins were analyzed by Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS). For glycerophospholipids (PC, PE, PI, PS, PG, PA) we applied direct infusion MS approaches (Shotgun Lipidomics).","INSTITUTE":"University of Cologne","DEPARTMENT":"Faculty of Medicine and University Hospital of Cologne, Cluster of Excellence Cellular Stress Responses in Aging-associated Diseases (CECAD)","LABORATORY":"CECAD Lipidomics/Metabolomics Facility","LAST_NAME":"Brodesser","FIRST_NAME":"Susanne","ADDRESS":"Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany","EMAIL":"susanne.brodesser@uk-koeln.de","PHONE":"+49 221 478 84015"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"S01_mitos_control.SMA_1",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"WT","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S02_mitos_control.SMA_2",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"WT","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S03_mitos_control.SMA_3",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"WT","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S04_mitos_control.SMA_4",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"WT","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S05_mitos_apoptosis.SMA_1",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"WT","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S06_mitos_apoptosis.SMA_2",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"WT","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S07_mitos_apoptosis.SMA_3",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"WT","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S08_mitos_apoptosis.SMA_4",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"WT","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S09_mitos_control_1",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S10_mitos_control_2",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S11_mitos_control_3",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S12_mitos_control_4",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S13_mitos_apoptosis_1",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S14_mitos_apoptosis_2",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S15_mitos_apoptosis_3",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S16_mitos_apoptosis_4",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S02_pulldown_Control_22.08",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"mEGFP-BAK","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S03_pulldown_Control_01.09",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"mEGFP-BAK","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S04_pulldown_Control_04.09",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"mEGFP-BAK","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S05_pulldown_Control_31.09",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"mEGFP-BAK","Condition":"control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S06_pulldown_Apoptosis_22.08",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"mEGFP-BAK","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S07_pulldown_Apoptosis_01.09",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"mEGFP-BAK","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S08_pulldown_Apoptosis_04.09",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"mEGFP-BAK","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S09_pulldown_Apoptosis_31.09",
"Factors":{"Sample source":"mitochondrial SMALPs","Genotype":"mEGFP-BAK","Condition":"apoptotic"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"S01_mitos_no.treatment_WT_1",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"untreated"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S01_mitos_no.treatment_WT_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S02_mitos_no.treatment_WT_2",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"untreated"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S02_mitos_no.treatment_WT_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S03_mitos_no.treatment_WT_3",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"untreated"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S03_mitos_no.treatment_WT_3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S04_mitos_no.treatment_WT_4",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"untreated"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S04_mitos_no.treatment_WT_4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S05_mitos_linoleic.acid_WT_1",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"linoleic acid"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S05_mitos_linoleic.acid_WT_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S06_mitos_linoleic.acid_WT_2",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"linoleic acid"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S06_mitos_linoleic.acid_WT_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S07_mitos_linoleic.acid_WT_3",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"linoleic acid"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S07_mitos_linoleic.acid_WT_3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S08_mitos_linoleic.acid_WT_4",
"Factors":{"Sample source":"total mitochondria","Genotype":"WT","Condition":"linoleic acid"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S08_mitos_linoleic.acid_WT_4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S09_smitos_no.treatment_KO_1",
"Factors":{"Sample source":"total mitochondria","Genotype":"FADS2 KO","Condition":"untreated"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S09_mitos_no.treatment_KO_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S10_mitos_no.treatment_KO_2",
"Factors":{"Sample source":"total mitochondria","Genotype":"FADS2 KO","Condition":"untreated"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S10_mitos_no.treatment_KO_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S11_mitos_no.treatment_KO_3",
"Factors":{"Sample source":"total mitochondria","Genotype":"FADS2 KO","Condition":"untreated"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S11_mitos_no.treatment_KO_3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S12_mitos_no.treatment_KO_4",
"Factors":{"Sample source":"total mitochondria","Genotype":"FADS2 KO","Condition":"untreated"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S12_mitos_no.treatment_KO_4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S13_mitos_linoleic.acid_KO_1",
"Factors":{"Sample source":"total mitochondria","Genotype":"FADS2 KO","Condition":"linoleic acid"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S13_mitos_linoleic.acid_KO_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S14_mitos_linoleic.acid_KO_2",
"Factors":{"Sample source":"total mitochondria","Genotype":"FADS2 KO","Condition":"linoleic acid"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S14_mitos_linoleic.acid_KO_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S15_mitos_linoleic.acid_KO_3",
"Factors":{"Sample source":"total mitochondria","Genotype":"FADS2 KO","Condition":"linoleic acid"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S15_mitos_linoleic.acid_KO_3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"S16_mitos_linoleic.acid_KO_4",
"Factors":{"Sample source":"total mitochondria","Genotype":"FADS2 KO","Condition":"linoleic acid"},
"Additional sample data":{"RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-","RAW_FILE_NAME":"FA_S16_mitos_linoleic.acid_KO_4.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Human osteosarcoma U2OS WT, U2OS BAK Ko expressing GFP BAK, and U2OS FADS2 KO cell lines were cultured at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen, Germany). For lipidomic experiments cells were incubated with 1 μM of ABT-737 and S63845 in the complete media and incubated for 50 min at 37°C and 5% CO2. FADS2 KO in U2OS cells was generated in the lab by the CRISPR/Cas9 method. Linoleic acid stock (50 mM) was prepared in ethanol and diluted into culture media before adding them to the cells. Mitochondria were isolated from cultured human osteosarcoma cells by mechanical disruption of cells followed by differential centrifugation: Cells were harvested by trypsinization, washed in PBS, and then resuspend in isolation buffer (IM;250 mM sucrose, 5 mM Tris, and 2 mM EDTA; pH 7.4 and protease inhibitor cocktail) and mechanically broken using glass homogenizer on ice (30-40 strokes on ice) and total cellular lysates were spin down first to remove nuclei and cell debris at 600 x g for 5 min and later at 10,800 x g for 10 min at 4°C to get the crude mitochondria. Mitochondrial pellet was washed 2-3 times with isolation buffer to remove other impurities from mitochondria. Isolated mitochondria were solubilized using SMA co-polymer. For this, mitochondria either from apoptotic or healthy cells were incubated with 0.5% SMA (2:1) for 45 min at room temperature with gentle rotation. Mitochondrial membrane was spun down at 100,000 x g for 40 min to separate solubilized SMALP from the insolubilized membrane. Next, the size of SMALP was analyzed by Dynamic Light Scattering (DLS). For DLS measurements, 15 μl of sample was added to a quartz cuvette which had been thoroughly cleaned with Milli-Q H2O. The cuvette was placed in DynaPro NanoStar (Wyatt Technology corporation, USA) and the sample was analyzed using 10 runs with 10 second acquisition time. This helps to determine the mass distribution of the sample as well as the estimated size of the particles. The distance distribution is shown on a log scale. The size of SMALP as well as the homogeneity with in the sample were also checked by Negative Transmission Electron Microscopy (TEM). For this the diluted SMALPs were placed onto a glow-discharged copper grid (Electron Microscopy Sciences) coated with a layer of thin carbon, washed twice with water, stained with 2% uranyl acetate for 5 min and then air-dried. The grids were imaged on a JEOL JEM2100PLUS electron microscope and recorded with a GATAN OneView camera (CECAD Imaging Facility). mEGFP-BAK-SMALPs were affinity purified from total solubilized mitochondrial membrane fraction (SMALP). For this total SMALP were incubated with 25 μl of GFP-trap MA beads for 90 min with slow rotation in cold room. Beads were washed 2 times with 100 μl of Tris buffer (50 mM Tris 150 mM NaCl pH 8), and finally resuspend in 100 ul of Tris buffer. Small aliquots of unbound and wash fractions were used to analyze the purification quality.","SAMPLE_TYPE":"Mitochondria"},

"TREATMENT":{"TREATMENT_SUMMARY":"The samples were not subjected to any further treatment."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Glycerophospholipids: Lipids from isolated mitochondria treated with or without SMA were extracted using a procedure previously described (Ejsing et al., 2009) with some modifications: 30-100 µl of sample were brought to a volume of 200 µl with 155 mM ammonium carbonate buffer. Lipids were extracted by adding 990 µl of chloroform/methanol 17:1 (v/v) and internal standards (125 pmol PC 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, 61 pmol PG 17:0/20:4, 72 pmol PA 17:0/20:4, 10 µl Cardiolipin Mix I; Avanti Polar Lipids), followed by shaking at 900 rpm/min in a ThermoMixer (Eppendorf) at 20 °C for 30 min. After centrifugation (12,000xg, 5 min, 4 °C), the lower (organic) phase was transferred to a new tube, and the upper phase was extracted again with 990 mL chloroform/methanol 2:1 (v/v). The combined organic phases were dried under a stream of nitrogen. The residues were resolved in 200 µl of methanol. Ceramides and sphingomyelins: For the analysis of ceramides and sphingomyelins in isolated mitochondria without and after SMA treatment, lipids were extracted as described above in the presence of 127 pmol ceramide 12:0 and 124 pmol sphingomyelin 12:0 (internal standards, Avanti Polar Lipids). The dried extracts were resolved in 100 µL of Milli-Q water and 750 µL of chloroform/methanol 1:2 (v/v). Alkaline hydrolysis of glycerolipids was conducted as previously published (Schwamb et al., 2012; Oteng et al., 2017). Fatty acids: To 100 µl of a suspension of isolated mitochondria in PBS, 500 µl of methanol, 250 µl of chloroform, and 0.5 µg palmitic-d31 acid (Sigma-Aldrich) as internal standard were added. The mixture was sonicated for 5 min, and lipids were extracted in a shaking bath at 48 °C for 1 h. Glycerolipids were degraded by alkaline hydrolysis adding 75 µl of 1 M potassium hydroxide in methanol. After 5 min of sonication, the extract was incubated for 1.5 h at 37 °C, and then neutralized with 6 µl of glacial acetic acid. 2 ml of chloroform and 4 ml of water were added to the extract which was vortexed vigorously for 30 sec and then centrifuged (4,000 × g, 5 min, 4 °C) to separate layers. The lower (organic) phase was transferred to a new tube, and the upper phase extracted with additional 2 ml of chloroform. The combined organic phases were dried under a stream of nitrogen. The residues were resolved in 200 µl of acetonitrile/water 2:1 (v/v) and sonicated for 5 min. After centrifugation (12,000 × g, 20 min, 4 °C), 40 µl of the clear supernatants were transferred to autoinjector vials. References: Ejsing et al., Proc Natl Acad Sci USA 2009, 106, 2136; Oteng et al., J Lipid Res 2017, 58, 1100; Schwamb et al., Blood 2012, 120, 3978."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Shimadzu Nexera X2","COLUMN_NAME":"Phenomenex Core-Shell Kinetex Biphenyl (100×3.0 mm, 2.6 μm, 100 Å)","SOLVENT_A":"100% water; 0.012 % acetic acid; 5 mM ammonium acetate","SOLVENT_B":"80% acetonitrile/20% isopropanol","FLOW_GRADIENT":"0 min: 55% B, 4 min: 95% B, 7 min: 95% B, 7.1 min: 55% B, 10 min: 55% B","FLOW_RATE":"0.7 ml/min","COLUMN_TEMPERATURE":"40"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"SCIEX QTRAP 6500","INSTRUMENT_TYPE":"QTRAP","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Fatty acid levels were determined by LC-ESI-MS/MS: Fatty acids were monitored in the negative ion mode using “pseudo” Multiple Reaction Monitoring (MRM) transitions (Hellmuth et al., Anal Chem 2012, 84, 1483). The instrument settings for nebulizer gas (Gas 1), turbo gas (Gas 2), curtain gas, and collision gas were 60 psi, 90 psi, 40 psi, and medium, respectively. The Turbo V ESI source temperature was 650 °C, and the ionspray voltage was -4 kV. The LC chromatogram peaks of the endogenous fatty acids and the internal standard palmitic-d31 acid were integrated using the MultiQuant 3.0.2 software (SCIEX)."},

"MS_METABOLITE_DATA":{
"Units":"counts per second (cps)",

"Data":[{"Metabolite":"Palmitic acid-d31 (IS)","S01_mitos_no.treatment_WT_1":"80585983","S02_mitos_no.treatment_WT_2":"152271276","S03_mitos_no.treatment_WT_3":"128426957","S04_mitos_no.treatment_WT_4":"140482152","S05_mitos_linoleic.acid_WT_1":"106544120","S06_mitos_linoleic.acid_WT_2":"78615892","S07_mitos_linoleic.acid_WT_3":"93275769","S08_mitos_linoleic.acid_WT_4":"114741724","S09_smitos_no.treatment_KO_1":"200014877","S10_mitos_no.treatment_KO_2":"156921007","S11_mitos_no.treatment_KO_3":"207400568","S12_mitos_no.treatment_KO_4":"203326103","S13_mitos_linoleic.acid_KO_1":"145157229","S14_mitos_linoleic.acid_KO_2":"118024938","S15_mitos_linoleic.acid_KO_3":"130704316","S16_mitos_linoleic.acid_KO_4":"108732056"},{"Metabolite":"Myristic acid 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"Metabolites":[{"Metabolite":"Palmitic acid-d31 (IS)","Q1 Mass (Da)":"286.1","Q3 Mass (Da)":"286.1","RT (min)":"2.19","CE (volts)":"-10","KEGG_ID":"N/A"},{"Metabolite":"Tetradecenoic acid (14:1)","Q1 Mass (Da)":"225.0","Q3 Mass (Da)":"225.0","RT (min)":"1.62","CE (volts)":"-8","KEGG_ID":"C08322"},{"Metabolite":"Myristic acid (14:0)","Q1 Mass (Da)":"227.0","Q3 Mass (Da)":"227.0","RT (min)":"1.84","CE (volts)":"-25","KEGG_ID":"C06424"},{"Metabolite":"Hexadecenoic acid (16:1)","Q1 Mass (Da)":"253.0","Q3 Mass (Da)":"253.0","RT (min)":"2.01","CE (volts)":"-30","KEGG_ID":"C21942/C08362"},{"Metabolite":"Palmitic acid (16:0)","Q1 Mass (Da)":"255.0","Q3 Mass (Da)":"255.0","RT (min)":"2.25","CE (volts)":"-35","KEGG_ID":"C00249"},{"Metabolite":"Octadecatrienoic acid (18:3)","Q1 Mass (Da)":"277.1","Q3 Mass (Da)":"277.1","RT (min)":"2.00","CE (volts)":"-10","KEGG_ID":"C06426/C08315/C08364/C06427"},{"Metabolite":"Octadecadienoic acid (18:2)","Q1 Mass (Da)":"279.0","Q3 Mass (Da)":"279.1","RT (min)":"2.20","CE (volts)":"-32","KEGG_ID":"C04056/C01595"},{"Metabolite":"Octadecenoic acid (18:1)","Q1 Mass (Da)":"281.1","Q3 Mass (Da)":"281.1","RT (min)":"2.42","CE (volts)":"-37","KEGG_ID":"C08363/C00712/C01712/C21944/C08367"},{"Metabolite":"Stearic acid (18:0)","Q1 Mass (Da)":"283.1","Q3 Mass (Da)":"283.1","RT (min)":"2.67","CE (volts)":"-35","KEGG_ID":"C01530"},{"Metabolite":"Arachidonic acid (20:4)","Q1 Mass (Da)":"303.1","Q3 Mass (Da)":"303.1","RT (min)":"2.20","CE (volts)":"-10","KEGG_ID":"C00219"},{"Metabolite":"Eicosatrienoic acid (20:3)","Q1 Mass (Da)":"305.1","Q3 Mass (Da)":"305.1","RT (min)":"2.38","CE (volts)":"-8","KEGG_ID":"C03242/C16522/C21938"},{"Metabolite":"Eicosadienoic acid (20:2)","Q1 Mass (Da)":"307.1","Q3 Mass (Da)":"307.2","RT (min)":"2.59","CE (volts)":"-8","KEGG_ID":"C16525/C21936"},{"Metabolite":"Eicosenoic acid (20:1)","Q1 Mass (Da)":"309.1","Q3 Mass (Da)":"309.1","RT (min)":"2.81","CE (volts)":"-8","KEGG_ID":"C16526/C21946"},{"Metabolite":"DHA (22:6)","Q1 Mass (Da)":"327.1","Q3 Mass (Da)":"327.0","RT (min)":"2.18","CE (volts)":"-6","KEGG_ID":"C06429"},{"Metabolite":"Docosadienoic acid (22:2)","Q1 Mass (Da)":"335.1","Q3 Mass (Da)":"335.1","RT (min)":"2.99","CE (volts)":"-8","KEGG_ID":"C16533"},{"Metabolite":"Docosenoic acid (22:1)","Q1 Mass (Da)":"337.1","Q3 Mass (Da)":"337.1","RT (min)":"3.19","CE (volts)":"-8","KEGG_ID":"C08316"}]
}

}