#METABOLOMICS WORKBENCH rcswasti_20250109_214156 DATATRACK_ID:5518 STUDY_ID:ST003660 ANALYSIS_ID:AN006011 PROJECT_ID:PR002270 VERSION 1 CREATED_ON January 14, 2025, 8:45 am #PROJECT PR:PROJECT_TITLE Kingdom-specific lipid unsaturation calibrates sequence evolution in membrane PR:PROJECT_TITLE arm subunits of eukaryotic respiratory complexes PR:PROJECT_SUMMARY Sequence evolution of protein complexes (PCs) is constrained by protein-protein PR:PROJECT_SUMMARY interactions (PPIs). PPI-interfaces are predominantly conserved and hotspots for PR:PROJECT_SUMMARY disease-related mutations. How do lipid-protein interactions (LPIs) constrain PR:PROJECT_SUMMARY sequence evolution of membrane-PCs? We explore Respiratory Complexes (RCs) as a PR:PROJECT_SUMMARY case study as these allow to compare sequence evolution in subunits exposed to PR:PROJECT_SUMMARY both lipids in inner-mitochondrial membrane (IMM) and lipid-free aqueous matrix. PR:PROJECT_SUMMARY We find that lipid-exposed surfaces of the IMM-subunits but not of the matrix PR:PROJECT_SUMMARY subunits are populated with non-PPI disease-causing mutations signifying LPIs in PR:PROJECT_SUMMARY stabilizing RCs. Further, IMM-subunits including their exposed surfaces show PR:PROJECT_SUMMARY high intra-kingdom sequence conservation but remarkably diverge beyond. PR:PROJECT_SUMMARY Molecular Dynamics simulation suggests contrasting LPIs of structurally PR:PROJECT_SUMMARY superimposable but sequence-wise diverged IMM-exposed helices of Complex I (CI) PR:PROJECT_SUMMARY subunit Ndufa1 from human and Arabidopsis depending on kingdom-specific PR:PROJECT_SUMMARY unsaturation of cardiolipin fatty acyl chains. in cellulo assays consolidate PR:PROJECT_SUMMARY inter-kingdom incompatibility of Ndufa1-helices due to the lipid-exposed amino PR:PROJECT_SUMMARY acids. Plant-specific unsaturated fatty acids in human cells also trigger PR:PROJECT_SUMMARY CI-instability. Taken together, we posit that altered LPIs calibrate sequence PR:PROJECT_SUMMARY evolution at the IMM-arms of eukaryotic RCs. PR:INSTITUTE CSIR-Centre for Cellular & Molecular Biology PR:LAST_NAME Raychaudhuri PR:FIRST_NAME Swasti PR:ADDRESS Habsiguda, Uppal Road, Hyderabad, 500007, Telangana, India PR:EMAIL rcswasti@ccmb.res.in PR:PHONE +91914027192558 #STUDY ST:STUDY_TITLE Kingdom-specific lipid unsaturation calibrates sequence evolution in membrane ST:STUDY_TITLE arm subunits of eukaryotic respiratory complexes ST:STUDY_SUMMARY Sequence evolution of protein complexes (PCs) is constrained by protein-protein ST:STUDY_SUMMARY interactions (PPIs). PPI-interfaces are predominantly conserved and hotspots for ST:STUDY_SUMMARY disease-related mutations. How do lipid-protein interactions (LPIs) constrain ST:STUDY_SUMMARY sequence evolution of membrane-PCs? We explore Respiratory Complexes (RCs) as a ST:STUDY_SUMMARY case study as these allow to compare sequence evolution in subunits exposed to ST:STUDY_SUMMARY both lipids in inner-mitochondrial membrane (IMM) and lipid-free aqueous matrix. ST:STUDY_SUMMARY We find that lipid-exposed surfaces of the IMM-subunits but not of the matrix ST:STUDY_SUMMARY subunits are populated with non-PPI disease-causing mutations signifying LPIs in ST:STUDY_SUMMARY stabilizing RCs. Further, IMM-subunits including their exposed surfaces show ST:STUDY_SUMMARY high intra-kingdom sequence conservation but remarkably diverge beyond. ST:STUDY_SUMMARY Molecular Dynamics simulation suggests contrasting LPIs of structurally ST:STUDY_SUMMARY superimposable but sequence-wise diverged IMM-exposed helices of Complex I (CI) ST:STUDY_SUMMARY subunit Ndufa1 from human and Arabidopsis depending on kingdom-specific ST:STUDY_SUMMARY unsaturation of cardiolipin fatty acyl chains. in cellulo assays consolidate ST:STUDY_SUMMARY inter-kingdom incompatibility of Ndufa1-helices due to the lipid-exposed amino ST:STUDY_SUMMARY acids. Plant-specific unsaturated fatty acids in human cells also trigger ST:STUDY_SUMMARY CI-instability. Taken together, we posit that altered LPIs calibrate sequence ST:STUDY_SUMMARY evolution at the IMM-arms of eukaryotic RCs. ST:INSTITUTE CSIR-Centre for Cellular & Molecular Biology ST:LAST_NAME Raychaudhuri ST:FIRST_NAME Swasti ST:ADDRESS Habsiguda, Uppal Road, Hyderabad, 500007, Telangana, India ST:EMAIL rcswasti@ccmb.res.in ST:PHONE +914027192558 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC SU:CELL_STRAIN_DETAILS HEK293T #FACTORS #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 - HEK293T-BSA-R1 Treatment:Control | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-BSA-R1.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-BSA-R2 Treatment:Control | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-BSA-R2.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-BSA-R3 Treatment:Control | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-BSA-R3.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-BSA-R4 Treatment:Control | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-BSA-R4.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-BSA-R5 Treatment:Control | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-BSA-R5.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-BSA-R6 Treatment:Control | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-BSA-R6.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 2-treated-R1 Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-2-R1.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 2-treated-R2 Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-2-R2.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 2-treated-R3 Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-2-R3.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 2-treated-R4 Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-2-R4.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 2-treated-R5 Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-2-R5.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 2-treated-R6 Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-2-R6.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 3-treated-R1 Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-3-R1.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 3-treated-R2 Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-3-R2.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 3-treated-R3 Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-3-R3.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 3-treated-R4 Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-3-R4.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 3-treated-R5 Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-3-R5.wiff SUBJECT_SAMPLE_FACTORS - HEK293T-18 3-treated-R6 Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells RAW_FILE_NAME(Raw data file name)=HEK-18-3-R6.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-1 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-1.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-2 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-2.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-3 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-3.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-4 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-4.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-5 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-5.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-6 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-6.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-7 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-7.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-8 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-8.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-9 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-9.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-10 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-10.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-11 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-11.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-12 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-12.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-13 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-13.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-14 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-14.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-15 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-15.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-16 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-16.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-17 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-17.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-18 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-18.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-19 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-19.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-20 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-20.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-21 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-21.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-22 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-22.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-23 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-23.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-24 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-24.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-25 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-25.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-26 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-26.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-27 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-27.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-28 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-28.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-29 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-29.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-30 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-30.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-31 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-31.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-32 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-32.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-33 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-33.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-34 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-34.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-35 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-35.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-36 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-36.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-37 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-37.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-38 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-38.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-39 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-39.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-40 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-40.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-41 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-41.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-42 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-42.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-43 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-43.wiff SUBJECT_SAMPLE_FACTORS - Experimental Blank-44 Treatment:Blank | Sample source:LC buffer RAW_FILE_NAME(Raw data file name)=Blank-44.wiff #COLLECTION CO:COLLECTION_SUMMARY HEK293T cells (Human Embryonic Kidney) were treated with BSA-conjugated linoleic CO:COLLECTION_SUMMARY acid and linolenic acid and incubated at 37°C for 72 hours in a CO2 incubator. CO:COLLECTION_SUMMARY Cells were trypsinized and pelleted, and mitochondria were isolated and stored CO:COLLECTION_SUMMARY at -80°C. Lipids were isolated, dried in a stream of nitrogen, and further CO:COLLECTION_SUMMARY stored at -80°C until further use CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Hs-A1-EGFP expressing HEK293T cells were treated with BSA-conjugated linoleic TR:TREATMENT_SUMMARY acid and linolenic acid as described by Omer et al (Oemer G, et al. The lipid TR:TREATMENT_SUMMARY environment modulates cardiolipin and phospholipid constitution in wild type and TR:TREATMENT_SUMMARY tafazzin-deficient cells. J Inherit Metab Dis 45, 38-50 (2022). PMID: 34494285) TR:TREATMENT_SUMMARY with slight modifications. Briefly, 10 mM solutions of fatty acids (Sigma) were TR:TREATMENT_SUMMARY prepared by dissolving in absolute ethanol. Meanwhile, 10% fatty acid-free BSA TR:TREATMENT_SUMMARY (Sigma) was prepared in 1X PBS (pH 7.4) and incubated at 55°C for 30 minutes TR:TREATMENT_SUMMARY with intermittent vortexing. The solution was cooled to 25°C and filter TR:TREATMENT_SUMMARY sterilized. The 10 mM fatty acid solution was diluted 1:20 in 10% BSA (final TR:TREATMENT_SUMMARY fatty acid concentration: 500 μM) and incubated at 55°C for 30 mins with TR:TREATMENT_SUMMARY intermittent vortexing. The BSA-conjugated fatty acid stock solution was further TR:TREATMENT_SUMMARY diluted 1:10 in incomplete DMEM (final concentration: 50 μM of fatty acid in 1% TR:TREATMENT_SUMMARY BSA) and supplemented to cells. Control cells were supplemented with TR:TREATMENT_SUMMARY BSA-conjugated ethanol. The cells were induced with 1mg/ml doxycycline and TR:TREATMENT_SUMMARY incubated at 37°C and 5 % CO2 for 72 hours. Prior to lipid supplementation, TR:TREATMENT_SUMMARY cells were plated in DMEM and 10% FBS and allowed to adhere for at least 16 TR:TREATMENT_SUMMARY hours. Cells were pelleted after 72 hours of treatment and mitochondria-enriched TR:TREATMENT_SUMMARY fraction preparation was performed according to Rawat et al. (Rawat S, Ghosh S, TR:TREATMENT_SUMMARY Mondal D, Anusha V, Raychaudhuri S. Increased supraorganization of respiratory TR:TREATMENT_SUMMARY complexes is a dynamic multistep remodelling in response to proteostasis stress. TR:TREATMENT_SUMMARY J Cell Sci 133, (2020). PMID: 32878939) with slight modification. Briefly, ∼20 TR:TREATMENT_SUMMARY million cells were homogenized at 4°C in a Dounce homogenizer with 600 ml of TR:TREATMENT_SUMMARY buffer A (83 mM sucrose, 10 mM MOPS pH 7.2). After adding an equal volume of TR:TREATMENT_SUMMARY buffer B (250 mM sucrose, 30 mM MOPS pH 7.2), nuclei and unbroken cells were TR:TREATMENT_SUMMARY removed by centrifugation at 1000 g for 5 min at 4°C. The supernatant was TR:TREATMENT_SUMMARY further centrifuged at 12,000 g for 5 min and washed twice with buffer C (320 mM TR:TREATMENT_SUMMARY sucrose, 1 mM EDTA, 10 mM Tris-HCl pH 7.4). The pellet was resuspended in 1X TR:TREATMENT_SUMMARY NativePAGE sample buffer (Invitrogen) to obtain a mitochondria-enriched fraction TR:TREATMENT_SUMMARY followed by storing at -80°C until further use. TR:TREATMENT BSA conjugated fatty acids TR:TREATMENT_COMPOUND Linoleic acid and Linolenic acid TR:TREATMENT_ROUTE Adding to cell culture media TR:TREATMENT_DOSE 50 μM TR:TREATMENT_DOSEDURATION 72 hours TR:TREATMENT_VEHICLE BSA TR:CELL_GROWTH_CONTAINER CO2 incubator TR:CELL_GROWTH_RATE Doubling time 16 hourse TR:CELL_MEDIA DMEM TR:CELL_ENVIR_COND 37°C, 5% CO2 TR:CELL_HARVESTING Trypnising and pelleting TR:CELL_PCT_CONFLUENCE 80% #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 150 mg of mitochondria isolated from BSA, linoleic and linolenic acid treated SP:SAMPLEPREP_SUMMARY Hs-A1-EGFP cells were resuspended in 250 μl of PBS. Lipids were isolated by SP:SAMPLEPREP_SUMMARY modified Folch method. Briefly 750 μl of 2:1 (v/v) chloroform (CHCl3): methanol SP:SAMPLEPREP_SUMMARY (MeOH) containing CL 56:0 (14:0)4 as an internal standard (IS) was added to the SP:SAMPLEPREP_SUMMARY mitochondrial fraction and vortexed thoroughly. Phase separation was achieved by SP:SAMPLEPREP_SUMMARY centrifugation at 2000 g for 5 mins. The organic phase was collected in a fresh SP:SAMPLEPREP_SUMMARY glass vial. Re-extraction was performed from the aqueous phase by adding 500 μl SP:SAMPLEPREP_SUMMARY of chloroform, 10% (v/v) formic acid, followed by vortex and centrifuge. The SP:SAMPLEPREP_SUMMARY organic phase was pooled and dried under the stream of nitrogen gas. Isolated SP:SAMPLEPREP_SUMMARY mitochondria and dried lipids were stored in -80°C until further use. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACTION_METHOD Modified Folch Method SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION CHCL2:MEOH- 1:2 SP:SAMPLE_SPIKING CL 56:0, cardiolipin SP:SUBCELLULAR_LOCATION Mitochondria #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Dried lipids were resuspended in 60 μl of 2:1 (v/v) dichloromethane (CH2Cl2): CH:CHROMATOGRAPHY_SUMMARY MeOH on the day of analysis. 25 μl of sample was analyzed using Shimadzu UHPLC CH:CHROMATOGRAPHY_SUMMARY LC40D coupled with Sciex QTRAP 6500+ mass spectrometer. Lipid extracts were CH:CHROMATOGRAPHY_SUMMARY separated on C18 reverse phase column (Phenomenex Gemini, 5 μm, 50 x 4.6 mm) CH:CHROMATOGRAPHY_SUMMARY using solvent A as 95:5 (v/v) H2O:MeOH + 0.1% Formic acid + 10mM ammonium CH:CHROMATOGRAPHY_SUMMARY formate and solvent B as 60:35:5 (v/v/v) Isopropanol:MeOH:H2O + 0.1% Formic acid CH:CHROMATOGRAPHY_SUMMARY + 10mM ammonium formate. LC gradient was set for 65 min: 5% B for 5 min with CH:CHROMATOGRAPHY_SUMMARY 0.5ml/min flow, a linear gradient of B from 5-100% over 25 min with 0.3ml/min CH:CHROMATOGRAPHY_SUMMARY flow, 100% B from 25-55 min with 0.3ml/min flow, 100% B from 55-60 min with CH:CHROMATOGRAPHY_SUMMARY 0.5ml/min flow and re-equilibration with 5% B for 5 min. The column was CH:CHROMATOGRAPHY_SUMMARY maintained at 55°C. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu UHPLC LC40D CH:COLUMN_NAME Phenomenex Gemini C18 (50 x 4.6mm,5um) CH:SOLVENT_A 95% water/5% methanol; 0.1% Formic acid; 10mM ammonium formate CH:SOLVENT_B 60% isopropanol/35% methanol/5% water; 0.1% Formic acid; 10mM ammonium formate CH:FLOW_GRADIENT 5% B for 5 min with 0.5ml/min flow, linear gradient of B from 5-100% over 25 min CH:FLOW_GRADIENT with 0.3ml/min flow, 100% B from 25-55 min with 0.3ml/min flow, 100% B from CH:FLOW_GRADIENT 55-60 min with 0.5ml/min flow and re-equilibration with 5% B for 5 min CH:FLOW_RATE 0.3mL/min-0.5mL/min CH:COLUMN_TEMPERATURE 55 CH:INJECTION_TEMPERATURE 4 CH:INTERNAL_STANDARD Cardiolipin 56:0 CH:SAMPLE_INJECTION 25μl CH:ANALYTICAL_TIME 65 mins CH:OVEN_TEMPERATURE 55°C CH:WASHING_BUFFER IsoP:MeOH:ACN:H2O- 1:1:1:1 #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE MS_protocol.txt AN:DATA_FORMAT .wiff #MS MS:INSTRUMENT_NAME ABI Sciex 6500+ QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Lipids were quantified using the scheduled multiple reaction monitoring (MRM) MS:MS_COMMENTS method in Sciex QTRAP 6500+ in high mass and negative ion mode using MS:MS_COMMENTS collision-induced dissociation (CID). 96 transitions from 45 CL species MS:MS_COMMENTS including internal standard (IS) were monitored. Source and gas settings: MS:MS_COMMENTS curtain gas pressure 35 A.U.; Collision Gas (CAD) High; Ionspray Voltage -4500V; MS:MS_COMMENTS temperature 550 °C; Ion source gas 1&2 55; declustering potential -80V; MS:MS_COMMENTS collision energy -120V; entrance potential -10V; exit potential -13V; MRM MS:MS_COMMENTS detection window of 45s for IS and 180s for other CLs; minimum dwell time 100ms. MS:MS_COMMENTS Multiple blanks were run between samples. The area under curve (AUC) was MS:MS_COMMENTS calculated using Sciex OS, Analytics version 3.3.1.43 software with a minimum MS:MS_COMMENTS peak width of 7 points while the rest of the parameters were default. A minimum MS:MS_COMMENTS of 3 transitions per CL species were monitored (Table 1, MS_protocol.txt) where MS:MS_COMMENTS one transition – -152.9 corresponded to the head group (Glycerolphosphate-H2O) MS:MS_COMMENTS and the other two transitions belonged to fatty acid chains. -152.9 was defined MS:MS_COMMENTS as a qualifier. CL species lacking qualifier peaks were not considered for MS:MS_COMMENTS analysis. Further, 2 transitions of fatty acids were considered as quantifiers, MS:MS_COMMENTS and CL species where both the transitions were identified and had AUC greater MS:MS_COMMENTS than or equal to the order of qualifier were only considered for analysis. Each MS:MS_COMMENTS spectra was subtracted with its respective blank. The AUC of each CL species was MS:MS_COMMENTS normalized and % Abundance was calculated using the formula provided in MS:MS_COMMENTS MS_protocol.txt MS:CAPILLARY_TEMPERATURE 550 °C MS:COLLISION_ENERGY -120V MS:COLLISION_GAS High MS:FRAGMENTATION_METHOD CID MS:ION_SOURCE_TEMPERATURE 55 MS:ION_SPRAY_VOLTAGE -4500V MS:MASS_ACCURACY 0.5Da MS:PRECURSOR_TYPE [M-H]- #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Blank substracted Area under curve MS_METABOLITE_DATA_START Samples HEK293T-BSA-R1 HEK293T-BSA-R2 HEK293T-BSA-R3 HEK293T-BSA-R4 HEK293T-BSA-R5 HEK293T-BSA-R6 HEK293T-18 2-treated-R1 HEK293T-18 2-treated-R2 HEK293T-18 2-treated-R3 HEK293T-18 2-treated-R4 HEK293T-18 2-treated-R5 HEK293T-18 2-treated-R6 HEK293T-18 3-treated-R1 HEK293T-18 3-treated-R2 HEK293T-18 3-treated-R3 HEK293T-18 3-treated-R4 HEK293T-18 3-treated-R5 HEK293T-18 3-treated-R6 Factors Treatment:Control | Sample source:HEK293T cultured cells Treatment:Control | Sample source:HEK293T cultured cells Treatment:Control | Sample source:HEK293T cultured cells Treatment:Control | Sample source:HEK293T cultured cells Treatment:Control | Sample source:HEK293T cultured cells Treatment:Control | Sample source:HEK293T cultured cells Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells Treatment:Linoleic acid treatment | Sample source:HEK293T cultured cells Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells Treatment:Linolenic acid treatment | Sample source:HEK293T cultured cells CL 16:1_16:1_16:1_16:1 184200 119200 177300 174000 48200 107700 2542 14250 12675 8055 3204 3102 13300 84240 23970 6615 9255 21880 CL 16:0_16:0_16:0_18:1 396 416794 0 0 55750 59300 71940 -10110 2210 0 30490 4698 2372 2418 0 0 861 27650 CL 16:0_16:1_16:1_18:2 46310 30270 31220 31990 20200 32540 2187 10950 5685 13390 11940 2705 8282 19580 8285 2457 7045 5228 CL 16:1_16:1_18:2_16:1 59830 75060 51460 53980 36460 39920 7515 28090 36310 17740 21300 4282 13490 37170 10850 2933 0 11740 CL 16:0_16:1_16:1_18:3 0 0 0 0 0 0 0 0 0 0 0 0 1520 25610 14010 0 0 4166 CL 16:0_16:0_18:1_18:1/ CL 16:0_16:1_18:0_18:1 -5827 -4306 5801 5812 341700 511000 13354 -16820 0 0 1131 -3848 -5364 -3336 0 0 172 -2348 CL 16:1_16:1_18:1_18:1 19610 12700 8003 -2169 0 25290 1704 3544 3848 6527 8810 0 0 0 291 0 3903 0 CL 16:0_16:0_18:1_18:3 0 0 0 3186 0 0 0 0 0 0 1135 0 1548 11340 0 0 0 0 CL 16:1_16:1_18:1_18:2/ CL 16:0_16:1_18:2_18:2 308700 248500 178300 230900 181600 178300 52250 75430 125678 83324 82910 20970 35210 158400 28080 15180 0 48210 CL 16:0_16:0_18:2_18:3/ CL 16:0_16:1_18:1_18:3/ CL 16:1_16:1_18:0_18:3 0 0 6549 7131 0 4633 4348 8415 10360 10910 0 0 12020 62040 26550 6253 13740 30500 CL 16:1_16:1_18:2_18:2 66650 102700 43500 46450 53540 67830 58730 159800 178800 107100 154100 23580 33250 61690 16990 9630 30840 41100 CL 16:1_16:1_18:1_18:3/ CL 16:0_16:1_18:2_18:3 4384 0 3061 0 0 0 0 0 0 0 0 0 44170 167300 63530 30960 64590 62970 CL 16:0_16:1_16:1_20:5 0 0 0 0 0 0 0 0 0 0 0 0 33500 114700 53010 13480 56960 55650 CL 16:1_16:1_18:2_18:3/ CL 16:0_16:1_18:3_18:3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CL 16:1_16:1_16:1_20:5 0 0 0 -954.6 0 0 0 0 0 0 0 0 37160 128900 82520 47430 66540 67900 CL 16:1_16:1_18:3_18:3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CL 16:0_18:1_18:1_18:1/ CL 16:1_18:0_18:1_18:1 149800 5340 70590 99120 63360 64800 0 5356 -2662 687 6364 3731 0 47460 8345 5952 0 10730 CL 16:0_18:0_18:0_18:3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CL 16:0_18:1_18:1_18:2 0 115000 0 0 6305 0 12060 19750 23900 0 0 6261 15080 4407 0 0 15320 2834 CL 16:1_18:0_18:0_18:3/ CL 16:0_18:0_18:1_18:3 0 0 0 0 0 0 0 0 0 0 0 0 2393 0 0 0 0 0 CL 16:1_18:1_18:1_18:2 453945 330200 191300 270356 290600 329300 33320 162500 89679 70508 93518 35180 7046 173700 30411 24480 5565 57250 CL 16:0_18:1_18:1_18:3/ CL 16:1_18:0_18:1_18:3 0 0 0 0 0 0 0 0 0 0 0 0 9817 14390 19300 2531 22550 22290 CL 16:1_18:1_18:2_18:2 201500 2683 109800 134500 250300 213600 258000 539000 496474 279900 473900 69930 0 181269 26555 25470 0 95140 CL 16:1_18:1_18:1_18:3/ CL 16:0_18:0_18:3_18:3 6451 3619 2661 3211 2756 3188 0 0 0 0 0 0 0 236500 64550 46530 8294 95430 CL 16:1_18:2_18:2_18:2 41140 86190 33100 29534 57110 59710 275600 623100 441100 326800 803400 63650 87750 235300 77084 44390 153300 126300 CL 16:1_18:1_18:2_18:3/ CL 16:1_18:0_18:3_18:3 0 0 0 0 0 0 0 0 0 0 0 0 67050 238400 91330 27250 154600 116100 CL 16:1_16:1_18:2_20:4 0 0 0 -2127 0 0 0 0 0 0 4357 0 96800 322000 158100 109700 370400 182300 CL 16:1_18:1_18:3_18:3 10900 0 3558 5592 0 0 0 0 7152 0 0 0 40080 70490 32740 22480 93810 48860 CL 16:1_16:1_18:2_20:5 0 0 0 0 0 0 0 0 0 0 0 0 67530 140100 51740 69040 255700 153100 CL 16:1_18:2_18:3_18:3 0 0 0 0 0 0 0 0 0 0 0 0 37280 72580 37460 29850 96980 66400 CL 16:1_18:3_18:3_18:3 0 0 0 0 0 0 0 0 0 0 2948 0 52190 131100 121200 107200 305400 160600 CL 18:1_18:1_18:1_18:1 167000 48950 53900 93180 86240 68460 6307 8482 3569 5880 4776 0 719.2 37920 6354 4978 324.6 9558 CL 18:1_18:1_18:1_18:2/ CL 18:0_18:1_18:2_18:2 121400 102600 43030 64450 125100 62440 19140 24580 20240 14130 31319 4777 4517 45450 9477 4271 7283 31540 CL 18:1_18:1_18:2_18:2 178200 278900 61060 96410 222800 191800 208300 246400 226614 162242 475487 54660 58170 98960 26154 9968 25250 49390 CL 18:1_18:1_18:1_18:3 0 0 0 0 0 0 0 0 0 0 0 0 4650 101800 17610 12650 16220 41360 CL 18:1_18:2_18:2_18:2 98615 137500 45260 45510 121800 113200 530800 171372.2 111600 466700 2344000 146200 -2595 204443 46340 33755 11015 143000 CL 18:1_18:1_18:2_18:3/ CL 18:0_18:1_18:3_18:3 0 0 0 0 1368 0 0 0 0 0 0 0 0 125000 27340 40230 0 35610 CL 18:2_18:2_18:2_18:2 0 34480 12120 16570 54220 51970 566800 890200 851900 355700 2517000 123100 81726 132191 40290 29930 -3449 121200 CL 18:1_18:1_18:3_18:3 0 0 0 -3064 0 1299 0 0 0 0 6643 0 0 273600 100000 77900 0 163100 CL 16:0_18:2_18:2_20:5/ CL 16:1_18:1_18:2_20:5 0 0 0 0 0 0 2626 0 0 0 6647 0 108000 203600 114400 65810 562300 183200 CL 18:2_18:2_18:2_18:3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CL 16:1_18:2_18:2_20:5 0 0 0 2809 0 0 0 0 0 0 11210 0 87200 169700 143100 141700 933500 282700 CL 18:2_18:2_18:3_18:3/ CL 18:1_18:3_18:3_18:3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CL 18:2_18:3_18:3_18:3 0 0 0 0 0 0 0 0 0 0 0 0 33480 70410 81760 40480 472100 203600 CL 14:0_14:0_14:0_14:0 262642000 204600000 504272000 691230000 365868000 431065000 806134000 427337000 387360000 636057000 298372000 380702000 749753000 765870000 555140000 469130000 806208000 697538000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pubchem Identifier Retention time (min) Quantified M/z (MS1) Quantified M/z (MS2) CL 16:1_16:1_16:1_16:1 131767780 35.4 1343.9 152.9, 253.2 CL 16:0_16:0_16:0_18:1 118701872 39.2 1377.9 152.9, 281.2, 255.2 CL 16:0_16:1_16:1_18:2 131766973 36.3 1371.9 152.9, 279.2, 253.2 CL 16:1_16:1_18:2_16:1 131767810 35.6 1369.9 152.9, 279.2, 253.2 CL 16:0_16:1_16:1_18:3 138284445 35.6 1369.9 152.9, 277.2, 253.2 CL 16:0_16:0_18:1_18:1/ CL 16:0_16:1_18:0_18:1 138125708/ 138179152 39.3 1403.9 152.9, 281.2, 255.2 CL 16:1_16:1_18:1_18:1 138272266 37 1399.9 152.9, 281.2, 253.2 CL 16:0_16:0_18:1_18:3 138292608 37 1399.9 152.9, 277.2, 281.2 CL 16:1_16:1_18:1_18:2/ CL 16:0_16:1_18:2_18:2 138195581/ 138148022 36.7 1397.9 152.9, 279.2, 253.2 CL 16:0_16:0_18:2_18:3/ CL 16:0_16:1_18:1_18:3/ CL 16:1_16:1_18:0_18:3 138127050/ 138230184/ 138205174 36.7 1397.9 152.9, 277.2, 253.2, 279.2 CL 16:1_16:1_18:2_18:2 138243780 36.1 1395.9 152.9, 279.2, 253.2 CL 16:1_16:1_18:1_18:3/ CL 16:0_16:1_18:2_18:3 138124283/ 138184666 36.1 1395.9 152.9, 277.2, 253.2, 279.2 CL 16:0_16:1_16:1_20:5 138260134 35.4 1393.9 152.9, 301.2, 253.2 CL 16:1_16:1_18:2_18:3/ CL 16:0_16:1_18:3_18:3 138204520/ 138223924 35.4 1393.9 152.9, 277.2, 253.2 CL 16:1_16:1_16:1_20:5 138121456 34.8 1391.9 152.9, 301.2, 253.2 CL 16:1_16:1_18:3_18:3 138138200 34.8 1391.9 152.9, 277.2, 253.2 CL 16:0_18:1_18:1_18:1/ CL 16:1_18:0_18:1_18:1 138189480/ 138181047 38.8 1430.1 152.9, 281.2, 255.2 CL 16:0_18:0_18:0_18:3 138262338 38.8 1430.1 152.9, 277.2, 255.2 CL 16:0_18:1_18:1_18:2 138120190 38.7 1427.9 152.9, 279.2, 281.2 CL 16:1_18:0_18:0_18:3/ CL 16:0_18:0_18:1_18:3 138289954/ 138256494 38.7 1427.9 152.9, 277.2, 281.2 CL 16:1_18:1_18:1_18:2 138255015 37.8 1425.9 152.9, 279.2, 281.2 CL 16:0_18:1_18:1_18:3/ CL 16:1_18:0_18:1_18:3 138191131/ 138305966 37.8 1425.9 152.9, 277.2, 281.2 CL 16:1_18:1_18:2_18:2 138209519 37 1423.9 152.9, 279.2, 281.2 CL 16:1_18:1_18:1_18:3/ CL 16:0_18:0_18:3_18:3 138236342/ 138311977 37 1423.9 152.9, 277.2, 281.2 CL 16:1_18:2_18:2_18:2 138126603 36.4 1421.9 152.9, 279.2, 253.2 CL 16:1_18:1_18:2_18:3/ CL 16:1_18:0_18:3_18:3 138223876/ 138225540 36.4 1421.9 152.9, 277.2, 253.2 CL 16:1_16:1_18:2_20:4 138184641 35.7 1419.9 152.9, 279.2, 253.2 CL 16:1_18:1_18:3_18:3 138261175 35.7 1419.9 152.9, 277.2, 253.2 CL 16:1_16:1_18:2_20:5 138205119 35.3 1417.9 152.9, 279.2, 253.2 CL 16:1_18:2_18:3_18:3 138156108 35.3 1417.9 152.9, 277.2, 253.2, 279.2 CL 16:1_18:3_18:3_18:3 138240999 34.6 1415.9 152.9, 277.2, 253.2 CL 18:1_18:1_18:1_18:1 10464221 40.3 1456.1 152.9, 281.2 CL 18:1_18:1_18:1_18:2/ CL 18:0_18:1_18:2_18:2 138173202/ 138283272 39.3 1454.1 152.9, 279.2, 281.2 CL 18:1_18:1_18:2_18:2 138243779 38.4 1451.9 152.9, 279.2, 281.2 CL 18:1_18:1_18:1_18:3 138129457 38.4 1451.9 152.9, 277.2, 281.2 CL 18:1_18:2_18:2_18:2 138291263 37.4 1449.9 152.9, 279.2, 281.2 CL 18:1_18:1_18:2_18:3/ CL 18:0_18:1_18:3_18:3 138146604/ 138304526 37.4 1449.9 152.9, 277.2, 281.2, 279.2 CL 18:2_18:2_18:2_18:2 138205444 36.7 1447.9 152.9, 279.2 CL 18:1_18:1_18:3_18:3 138164840 36.7 1447.9 152.9, 277.2, 281.2 CL 16:0_18:2_18:2_20:5/ CL 16:1_18:1_18:2_20:5 138279561/ 138138816 36.1 1445.9 152.9, 301.2, 279.2 CL 18:2_18:2_18:2_18:3 138293711 36.1 1445.9 152.9, 277.2, 279.2 CL 16:1_18:2_18:2_20:5 138173105 35.5 1443.9 152.9, 301.2, 279.2 CL 18:2_18:2_18:3_18:3/ CL 18:1_18:3_18:3_18:3 138157089/ 138184228 35.5 1443.9 152.9, 277.2, 279.2 CL 18:2_18:3_18:3_18:3 138158086 34.8 1441.9 152.9, 277.2, 279.2 CL 14:0_14:0_14:0_14:0 131797799 34.2 1239.9 152.9, 227.2 METABOLITES_END #END