#METABOLOMICS WORKBENCH Brodesser_20230816_054636 DATATRACK_ID:4224 STUDY_ID:ST003114 ANALYSIS_ID:AN005105 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                   	Phenomenex Core-Shell Kinetex Biphenyl (100×3.0 mm, 2.6 μm, 100 Å)
CH:SOLVENT_A                     	100% water; 0.012 % acetic acid; 5 mM ammonium acetate
CH:SOLVENT_B                     	80% acetonitrile/20% isopropanol
CH:FLOW_GRADIENT                 	0 min: 55% B, 4 min: 95% B, 7 min: 95% B, 7.1 min: 55% B, 10 min: 55% B
CH:FLOW_RATE                     	0.7 ml/min
CH:COLUMN_TEMPERATURE            	40
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	SCIEX QTRAP 6500
MS:INSTRUMENT_TYPE               	QTRAP
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Fatty acid levels were determined by LC-ESI-MS/MS: Fatty acids were monitored in
MS:MS_COMMENTS                   	the negative ion mode using “pseudo” Multiple Reaction Monitoring (MRM)
MS:MS_COMMENTS                   	transitions (Hellmuth et al., Anal Chem 2012, 84, 1483). The instrument settings
MS:MS_COMMENTS                   	for nebulizer gas (Gas 1), turbo gas (Gas 2), curtain gas, and collision gas
MS:MS_COMMENTS                   	were 60 psi, 90 psi, 40 psi, and medium, respectively. The Turbo V ESI source
MS:MS_COMMENTS                   	temperature was 650 °C, and the ionspray voltage was -4 kV. The LC chromatogram
MS:MS_COMMENTS                   	peaks of the endogenous fatty acids and the internal standard palmitic-d31 acid
MS:MS_COMMENTS                   	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_no.treatment_WT_1	S02_mitos_no.treatment_WT_2	S03_mitos_no.treatment_WT_3	S04_mitos_no.treatment_WT_4	S05_mitos_linoleic.acid_WT_1	S06_mitos_linoleic.acid_WT_2	S07_mitos_linoleic.acid_WT_3	S08_mitos_linoleic.acid_WT_4	S09_smitos_no.treatment_KO_1	S10_mitos_no.treatment_KO_2	S11_mitos_no.treatment_KO_3	S12_mitos_no.treatment_KO_4	S13_mitos_linoleic.acid_KO_1	S14_mitos_linoleic.acid_KO_2	S15_mitos_linoleic.acid_KO_3	S16_mitos_linoleic.acid_KO_4
Factors	Sample source:total mitochondria | Genotype:WT | Condition:untreated	Sample source:total mitochondria | Genotype:WT | Condition:untreated	Sample source:total mitochondria | Genotype:WT | Condition:untreated	Sample source:total mitochondria | Genotype:WT | Condition:untreated	Sample source:total mitochondria | Genotype:WT | Condition:linoleic acid	Sample source:total mitochondria | Genotype:WT | Condition:linoleic acid	Sample source:total mitochondria | Genotype:WT | Condition:linoleic acid	Sample source:total mitochondria | Genotype:WT | Condition:linoleic acid	Sample source:total mitochondria | Genotype:FADS2 KO | Condition:untreated	Sample source:total mitochondria | Genotype:FADS2 KO | Condition:untreated	Sample source:total mitochondria | Genotype:FADS2 KO | Condition:untreated	Sample source:total mitochondria | Genotype:FADS2 KO | Condition:untreated	Sample source:total mitochondria | Genotype:FADS2 KO | Condition:linoleic acid	Sample source:total mitochondria | Genotype:FADS2 KO | Condition:linoleic acid	Sample source:total mitochondria | Genotype:FADS2 KO | Condition:linoleic acid	Sample source:total mitochondria | Genotype:FADS2 KO | Condition:linoleic acid
Palmitic acid-d31 (IS)	80585983	152271276	128426957	140482152	106544120	78615892	93275769	114741724	200014877	156921007	207400568	203326103	145157229	118024938	130704316	108732056
Myristic acid (14:0)	106821370	244677334	116764012	132615826	220350427	102707955	150567176	200451789	77541872	136571484	97412177	143373509	112732224	117896785	138868645	105025430
Tetradecenoic acid (14:1)	27199178	72131758	27435856	30558515	34138804	15029864	24131881	31265239	3614802	5932098	5205180	7224716	4393164	5583191	6017350	5487082
Palmitic acid (16:0)	24491134	37693196	28509507	29437283	32344318	23263138	27689886	30987833	24595534	24296073	28269110	28578433	24399895	26592246	24170518	25934787
Hexadecenoic acid (16:1)	90228905	172966565	95171389	104664650	112500184	59878382	81731668	105395926	51069089	76269327	74734373	70914975	52217357	67864507	71173406	61940172
Stearic acid (18:0)	76419479	132629534	92937391	100050129	124668216	76005966	95963121	115313639	67385704	60942398	85167923	82196163	75566653	87453070	76205240	80462974
Octadecenoic acid (18:1)	32147406	47563727	33440782	36835342	36020496	26120891	31707955	37288204	30910108	38687023	39492535	38060870	30146170	34473821	36929241	32645677
Octadecadienoic acid (18:2)	16789690	29055158	15123200	17084808	68332531	39406124	47448521	61365253	9393230	13971254	13114640	12505715	68206075	86964807	97064094	82978060
Octadecatrienoic acid (18:3)	36160015	86896917	32701389	40342702	334577957	139276139	199396744	300711902	6981400	10587814	11114985	10514718	9308828	13455099	13670712	11527654
Eicosenoic acid (20:1)	166124914	383320389	192277743	218536692	217235893	102730703	157512945	227346118	176182421	259935474	277151046	255389181	197798562	231816041	250292880	209968590
Eicosadienoic acid (20:2)	233514901	464157978	241044674	280555729	321728909	164563210	225508119	305840526	98395041	167111269	169201006	158807471	276962370	363995420	401509667	346055013
Eicosatrienoic acid (20:3)	410614961	683328458	415085720	470095757	659649897	455076940	545730342	638782448	71306944	103852926	105555583	103717508	168981875	214498836	227348393	193916867
Arachidonic acid (20:4)	389643000	631560227	422181091	470596776	746333384	520642388	608536516	729890832	275501273	365338558	375091363	355825148	296595005	334711375	370509099	305804067
Docosenoic acid (22:1)	78966085	246505457	94898371	108407405	138062974	53419373	79912824	147048231	102771429	131128251	151754367	142505304	105003251	129349914	148723961	112534448
Docosadienoic acid (22:2)	45668543	161116848	50377755	62157663	101681828	32331339	56402714	89983655	49169547	86727383	86187135	78292922	101327259	146036922	164120467	128564669
DHA (22:6)	229508492	423795788	246409595	276763750	203650120	129980947	169560130	220153677	126748952	190141217	193241652	184225188	106657972	133266520	146361018	118718201
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Q1 Mass (Da)	Q3 Mass (Da)	RT (min)	CE (volts)	KEGG_ID
Palmitic acid-d31 (IS)	286.1	286.1	2.19	-10	N/A
Tetradecenoic acid (14:1)	225.0	225.0	1.62	-8	C08322
Myristic acid (14:0)	227.0	227.0	1.84	-25	C06424
Hexadecenoic acid (16:1)	253.0	253.0	2.01	-30	C21942/C08362
Palmitic acid (16:0)	255.0	255.0	2.25	-35	C00249
Octadecatrienoic acid (18:3)	277.1	277.1	2.00	-10	C06426/C08315/C08364/C06427
Octadecadienoic acid (18:2)	279.0	279.1	2.20	-32	C04056/C01595
Octadecenoic acid (18:1)	281.1	281.1	2.42	-37	C08363/C00712/C01712/C21944/C08367
Stearic acid (18:0)	283.1	283.1	2.67	-35	C01530
Arachidonic acid (20:4)	303.1	303.1	2.20	-10	C00219
Eicosatrienoic acid (20:3)	305.1	305.1	2.38	-8	C03242/C16522/C21938
Eicosadienoic acid (20:2)	307.1	307.2	2.59	-8	C16525/C21936
Eicosenoic acid (20:1)	309.1	309.1	2.81	-8	C16526/C21946
DHA (22:6)	327.1	327.0	2.18	-6	C06429
Docosadienoic acid (22:2)	335.1	335.1	2.99	-8	C16533
Docosenoic acid (22:1)	337.1	337.1	3.19	-8	C08316
METABOLITES_END
#END