#METABOLOMICS WORKBENCH Brodesser_20230816_054636 DATATRACK_ID:4224 STUDY_ID:ST003114 ANALYSIS_ID:AN005103 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_SUMMARY        	Note: Macherey-Nagel does not offer this column as standard, but manufactures it
CH:CHROMATOGRAPHY_SUMMARY        	on request.
CH:CHROMATOGRAPHY_TYPE           	Normal phase
CH:INSTRUMENT_NAME               	Agilent 1260
CH:COLUMN_NAME                   	Macherey-Nagel Nucleosil NH2 (50×2 mm, 3 µm, 120 Å)
CH:SOLVENT_A                     	97% acetonitrile/2% methanol/1% acetic acid; 5 mM ammonium acetate
CH:SOLVENT_B                     	99% methanol/1% acetic acid; 5 mM ammonium acetate
CH:FLOW_GRADIENT                 	0 min: 0% B, 0.5 min: 0% B, 0.7 min: 10% B, 1.2 min: 10% B, 1.6 min: 18% B, 2.2
CH:FLOW_GRADIENT                 	min: 18% B, 2.6 min: 100% B, 4.5 min: 100% B, 4.9 min: 0% B, 6.5 min: 0% B
CH:FLOW_RATE                     	0.75 ml/min
CH:COLUMN_TEMPERATURE            	20
#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                   	LC-ESI-MS/MS analysis of ceramides and sphingomyelins was conducted as
MS:MS_COMMENTS                   	previously published (Oteng et al., J Lipid Res 2017, 58, 1100; Schwamb et al.,
MS:MS_COMMENTS                   	Blood 2012, 120, 3978).
#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	S02_pulldown_Control_22.08	S03_pulldown_Control_01.09	S04_pulldown_Control_04.09	S05_pulldown_Control_31.09	S06_pulldown_Apoptosis_22.08	S07_pulldown_Apoptosis_01.09	S08_pulldown_Apoptosis_04.09	S09_pulldown_Apoptosis_31.09
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	Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:control	Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:control	Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:control	Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:control	Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:apoptotic	Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:apoptotic	Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:apoptotic	Sample source:mitochondrial SMALPs | Genotype:mEGFP-BAK | Condition:apoptotic
Cer 18:1;O2/12:0 (IS)	62854554	66209758	76873410	62776237	66938341	70040240	69846020	55922546	60251711	42608445	64366517	76913545	68460397	65026514	56585330	53587576	107295534	76440194	55636065	20219126	53473727	35729833	28589720	45754100
Cer 18:1;O2/14:0	166415	208355	189673	85612	241639	238379	187905	262696	108619	42847	833284	189174	625663	833130	575225	922375	54628	60939	70284	20596	80650	40781	57245	58972
Cer 18:1;O2/16:0	707079	525720	690265	475657	943034	556643	736912	859080	1514192	287945	5020098	2323094	3599368	2254612	4836885	8097698	247019	195254	260178	117059	132863	124388	106748	96899
Cer 18:1;O2/16:1	289452	332987	438022	265762	317874	320797	328156	360552	275592	130881	622463	426974	700192	601219	595320	813611	217817	139110	145039	86629	137624	77460	78342	103079
Cer 18:1;O2/18:0	93010	107007	119940	82956	107019	101993	100859	102582	146936	50274	344456	216836	262172	177972	176149	169199	75767	53411	46076	37775	36005	27653	27684	27707
Cer 18:1;O2/18:1	29327	29014	47621	24465	36606	33880	46105	86331	42774	27466	274240	99960	242440	239012	267747	427941	51996	11806	101465	11690	24882	39461	41404	22650
Cer 18:1;O2/20:0	22718	24718	46116	21250	31901	29923	46415	44691	30617	17747	162891	59629	67937	113359	171829	259566	35982	25331	83529	17608	37049	49150	70862	24721
Cer 18:1;O2/22:0	53712	61803	63359	45166	80599	68236	73786	82794	103043	19376	279595	160076	195535	129368	208687	241106	30483	29338	24664	16673	17727	13579	12779	12359
Cer 18:1;O2/22:1	11014	9410	11667	8575	16362	13486	12635	15043	15588	5762	41525	24694	38205	18043	20130	19403	7500	5179	5371	4541	3759	3121	3853	2391
Cer 18:1;O2/24:0	187281	226426	296324	200302	313191	230086	297621	308867	419198	42552	1148856	656941	504490	318797	452145	227250	94265	76360	64128	32368	64401	64696	43775	29789
Cer 18:1;O2/24:1	279410	258716	323549	225698	450891	308540	360553	396209	588662	112780	1692830	945729	806230	421998	514107	332136	81002	72089	68419	26702	56178	58041	54028	31800
Cer 18:1;O2/26:0	24565	44345	45400	24399	33665	37397	37108	32777	28729	22835	86587	50959	105102	31845	42370	32021	24906	22067	15368	15627	14382	8135	12982	29284
Cer 18:1;O2/26:1	38241	34264	41480	29396	59842	35117	42629	43848	63750	11172	182126	99098	133229	49789	63645	47075	20883	13299	13715	7619	13277	11260	10399	7766
SM 18:1;O2/12:0 (IS)	12278792	8012135	9560593	7234260	10909370	9227934	8042176	8749546	5192979	4388543	7294405	4783978	3249252	5459398	3762596	4763562	6031436	2810836	3941971	1920769	2293461	2767661	1995166	2167435
SM 18:1;O2/14:0	94606	88883	97895	75260	96677	109968	97544	123902	521547	218511	1491490	442204	276249	391301	275626	294585	21415	6285	18044	6028	9765	7941	7990	7464
SM 18:1;O2/16:0	1044344	1008021	1212627	834530	1063265	1237416	1053088	1250897	6031564	2560988	16194169	5372633	3290884	4498409	3336856	3452847	389702	152442	245607	126936	147996	153156	161516	154746
SM 18:1;O2/16:1	135962	142415	186793	119620	149586	186110	170172	191336	763525	303735	2398036	691627	432825	552671	402177	407760	27405	7243	18256	8005	15089	8657	13470	7519
SM 18:1;O2/18:0	98812	71843	82240	62439	83545	87095	74666	88364	193565	96396	496849	171180	113825	175555	125032	136740	62013	26380	39298	18462	19894	22488	20273	19994
SM 18:1;O2/18:1	19778	21898	20443	17934	19555	23781	16943	25092	87686	35811	258691	75775	50481	69176	50008	48866	10872	6004	6245	2119	2866	2010	3230	1758
SM 18:1;O2/20:0	11950	11006	13824	14339	11565	14205	20162	20171	57791	23703	188498	45994	31681	45782	46420	35841	9121	7809	8264	7643	4029	3150	3742	3765
SM 18:1;O2/22:0	19645	16031	24899	26856	22605	23994	27927	27339	108638	40384	288152	81887	50841	116251	82936	72392	16353	17437	10521	11950	5891	6095	6293	7965
SM 18:1;O2/22:1	17822	19579	21952	18626	19923	25394	23006	26170	88713	34253	247953	76712	45124	73197	52106	47383	5597	2363	3951	1843	2727	1679	2817	1881
SM 18:1;O2/24:0	25741	31007	47903	45112	33838	51633	46302	46632	261345	92297	667979	208439	120022	223134	180211	162189	15274	7398	11608	4912	9390	5353	6031	5704
SM 18:1;O2/24:1	126286	137234	178388	128190	135651	201226	161674	187597	790747	312198	2093246	688227	432088	604605	436120	430516	33083	13053	25564	8287	18209	9612	14881	9439
SM 18:1;O2/26:0	2641	1953	3475	3172	3212	3322	3803	3636	11973	4405	31766	8872	5933	11287	9460	7454	753	248	1117	577	635	657	361	490
SM 18:1;O2/26:1	5464	4928	7479	5617	6445	7777	7781	8506	30601	11073	75934	24921	17729	28354	18498	17424	2471	1205	1717	629	1594	871	998	783
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Q1 Mass (Da)	Q3 Mass (Da)	RT (min)	CE (volts)	PubChem_CID	LM_ID
Cer 18:1;O2/12:0 (IS)	482.6	264.4	0.32	35	5283562	LMSP02010002
Cer 18:1;O2/14:0	510.6	264.4	0.32	36	5282310	LMSP02010001
Cer 18:1;O2/16:0	538.7	264.4	0.32	37	5283564	LMSP02040003
Cer 18:1;O2/16:1	536.7	264.4	0.32	37	N/A	N/A
Cer 18:1;O2/18:0	566.7	264.4	0.32	39	5283565	LMSP02010006
Cer 18:1;O2/18:1	564.7	264.4	0.32	39	5283563	LMSP02010003
Cer 18:1;O2/20:0	594.7	264.4	0.32	41	5283566	LMSP02010007
Cer 18:1;O2/22:0	622.8	264.4	0.32	41	5283567	LMSP02010008
Cer 18:1;O2/22:1	620.8	264.4	0.32	41	N/A	N/A
Cer 18:1;O2/24:0	650.9	264.4	0.32	43	5283571	LMSP02010012
Cer 18:1;O2/24:1	648.9	264.4	0.32	43	5283568	LMSP02010009
Cer 18:1;O2/26:0	678.9	264.4	0.32	45	5283570	LMSP02010011
Cer 18:1;O2/26:1	676.9	264.4	0.32	45	5283569	LMSP02010010
SM 18:1;O2/12:0 (IS)	647.7	184.4	4.17	39	44260123	LMSP03010002
SM 18:1;O2/14:0	675.7	184.4	4.16	40	11433862	LMSP03010028
SM 18:1;O2/16:0	703.8	184.4	4.15	41	9939941	LMSP03010003
SM 18:1;O2/16:1	701.8	184.4	4.15	41	52931145	LMSP03010041
SM 18:1;O2/18:0	731.8	184.4	4.13	43	6453725	LMSP03010001
SM 18:1;O2/18:1	729.8	184.4	4.13	43	6443882	LMSP03010029
SM 18:1;O2/20:0	759.8	184.4	4.13	44	44260124	LMSP03010005
SM 18:1;O2/22:0	787.9	184.4	4.12	44	44260125	LMSP03010006
SM 18:1;O2/22:1	785.9	184.4	4.12	44	52931203	LMSP03010072
SM 18:1;O2/24:0	815.9	184.4	4.11	45	44260127	LMSP03010008
SM 18:1;O2/24:1	813.9	184.4	4.11	45	44260126	LMSP03010007
SM 18:1;O2/26:0	843.9	184.4	4.09	47	44260129	LMSP03010010
SM 18:1;O2/26:1	841.9	184.4	4.09	47	44260128	LMSP03010009
METABOLITES_END
#END