#METABOLOMICS WORKBENCH Kugan91_20250529_173921 DATATRACK_ID:5968 STUDY_ID:ST003960 ANALYSIS_ID:AN006508 PROJECT_ID:PR002482 VERSION 1 CREATED_ON June 1, 2025, 5:31 pm #PROJECT PR:PROJECT_TITLE Complex II assembly drives metabolic adaptation to OXPHOS dysfunction. PR:PROJECT_SUMMARY During acute oxidative phosphorylation (OXPHOS) dysfunction, the reverse PR:PROJECT_SUMMARY activity of succinate dehydrogenase (Complex II) maintains the redox state of PR:PROJECT_SUMMARY Coenzyme Q by utilizing either fumarate or oxygen as terminal electron PR:PROJECT_SUMMARY acceptors. The tendency for one over another has been suggested to be PR:PROJECT_SUMMARY tissue-specific, but the underlying mechanism and consequence of this is PR:PROJECT_SUMMARY unknown. Using quantitative proteomics to screen a panel of HEK293T knockout PR:PROJECT_SUMMARY cell lines, we identified an increase in SDHAF2 protein, a Complex II assembly PR:PROJECT_SUMMARY factor that enhances the flavination of catalytic subunit SDHA, as critical for PR:PROJECT_SUMMARY metabolic adaptation during OXPHOS stress in HEK293T cells. Loss of SDHAF2 PR:PROJECT_SUMMARY during Complex III inhibition resulted in a reduction in Complex II F-site PR:PROJECT_SUMMARY derived reactive oxygen species (ROS), a severe growth impairment, and a net PR:PROJECT_SUMMARY reductive TCA cycle driven by an inability of mitochondria to support additional PR:PROJECT_SUMMARY Complex II assembly. This in turn leads to use of fumarate as terminal electron PR:PROJECT_SUMMARY acceptor at the cost of a ROS-mediated switch to glycolysis. Cell lines adapted PR:PROJECT_SUMMARY to glycolysis did not accumulate SDHAF2 upon OXPHOS stress and exhibited a net PR:PROJECT_SUMMARY reductive TCA cycle and mild growth phenotypes with or without SDHAF2 being PR:PROJECT_SUMMARY present. Thus, our study reveals how Complex II assembly controls a balance PR:PROJECT_SUMMARY between protection of the Q-pool and ROS-meditated signaling during oxidative PR:PROJECT_SUMMARY stress in cells reliant on mitochondrial OXPHOS. PR:INSTITUTE University of Melbourne PR:LAST_NAME Roopasingam PR:FIRST_NAME Kugapreethan PR:ADDRESS 30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, PR:ADDRESS University of Melbourne, Parkville, VIC, Australia. PR:EMAIL k.roopasingam@unimelb.edu.au PR:PHONE 0434297212 #STUDY ST:STUDY_TITLE Relative quantification of lactic acid secreted in media from cell lines treated ST:STUDY_TITLE with or without Antimycin A. ST:STUDY_SUMMARY This study aimed to investigate the lactate secretion as a measurement of ST:STUDY_SUMMARY glycolytic switch in subjected cell lines during OXPHOS dysfunction using ST:STUDY_SUMMARY targeted GC-MS analysis. ST:INSTITUTE University of Melbourne ST:LAST_NAME Roopasingam ST:FIRST_NAME Kugapreethan ST:ADDRESS 30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, ST:ADDRESS University of Melbourne, Parkville, VIC, Australia. ST:EMAIL k.roopasingam@unimelb.edu.au ST:PHONE 0434297212 #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 - 1-1_Fresh Media Sample source:1-1_Fresh Media_001 | Treatment:Control neg RAW_FILE_NAME(Raw file name)=1-1_Fresh Media_UNK-0012_2052025_12.qgd SUBJECT_SAMPLE_FACTORS - 1-2_Fresh Media Sample source:1-1_Fresh Media_002 | Treatment:Control neg RAW_FILE_NAME(Raw file name)=1-2_Fresh Media_UNK-0031_2052025_31.qgd SUBJECT_SAMPLE_FACTORS - 1-3_Fresh Media Sample source:1-1_Fresh Media_003 | Treatment:Control neg RAW_FILE_NAME(Raw file name)=1-3_Fresh Media_UNK-0024_2052025_24.qgd SUBJECT_SAMPLE_FACTORS - 2-1_HEKWT Sample source:2-1_HEKWT_001 | Treatment:Control RAW_FILE_NAME(Raw file name)=2-1_HEKWT_UNK-0015_2052025_15.qgd SUBJECT_SAMPLE_FACTORS - 2-2_HEKWT Sample source:2-1_HEKWT_002 | Treatment:Control RAW_FILE_NAME(Raw file name)=2-2_HEKWT_UNK-0023_2052025_23.qgd SUBJECT_SAMPLE_FACTORS - 2-3_HEKWT Sample source:2-1_HEKWT_003 | Treatment:Control RAW_FILE_NAME(Raw file name)=2-3_HEKWT_UNK-0008_2052025_8.qgd SUBJECT_SAMPLE_FACTORS - 3-1_HEKWT_A-A Sample source:3-1_HEKWT_A-A_001 | Treatment:Treatment RAW_FILE_NAME(Raw file name)=3-1_HEKWT_A-A_UNK-0022_2052025_22.qgd SUBJECT_SAMPLE_FACTORS - 3-2_HEKWT_A-A Sample source:3-1_HEKWT_A-A_002 | Treatment:Treatment RAW_FILE_NAME(Raw file name)=3-2_HEKWT_A-A_UNK-0025_2052025_25.qgd SUBJECT_SAMPLE_FACTORS - 3-3_HEKWT_A-A Sample source:3-1_HEKWT_A-A_003 | Treatment:Treatment RAW_FILE_NAME(Raw file name)=3-3_HEKWT_A-A_UNK-0019_2052025_19.qgd SUBJECT_SAMPLE_FACTORS - 4-1_HEKSDHAF2_A-A Sample source:4-1_HEKSDHAF2_A-A_001 | Treatment:Treatment RAW_FILE_NAME(Raw file name)=4-1_HEKSDHAF2_A-A_UNK-0009_2052025_9.qgd SUBJECT_SAMPLE_FACTORS - 4-2_HEKSDHAF2_A-A Sample source:4-1_HEKSDHAF2_A-A_002 | Treatment:Treatment RAW_FILE_NAME(Raw file name)=4-2_HEKSDHAF2_A-A_UNK-0029_2052025_29.qgd SUBJECT_SAMPLE_FACTORS - 4-3_HEKSDHAF2_A-A Sample source:4-1_HEKSDHAF2_A-A_003 | Treatment:Treatment RAW_FILE_NAME(Raw file name)=4-3_HEKSDHAF2_A-A_UNK-0014_2052025_14.qgd #COLLECTION CO:COLLECTION_SUMMARY Cells were seeded at 50% confluency in 6-well dishes containing DMEM media CO:COLLECTION_SUMMARY (Thermo Scientific) 24 hours prior to the experiment. For metabolite extraction, CO:COLLECTION_SUMMARY 50 ul of media was collected from wells with and without the cells. CO:SAMPLE_TYPE Media #TREATMENT TR:TREATMENT_SUMMARY The drug treatment was performed for 8 hours prior to media collection and the TR:TREATMENT_SUMMARY collected media was snap-frozen before polar metabolite extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A monophasic extraction protocol was used to extract lactic acid from the media. SP:SAMPLEPREP_SUMMARY To 30 µL of media, 90 µL of 100% MeOH containing 1.2 nmol of 13C515N valine SP:SAMPLEPREP_SUMMARY and 1.2 nmol of 13C6 sorbitol was added. Each sample was vortexed and then SP:SAMPLEPREP_SUMMARY incubated at 4˚C for 10 min with continuous agitation (12 g) using an Eppendorf SP:SAMPLEPREP_SUMMARY Thermomixer C. The samples were centrifuged at 4˚C for 10 min at 16000 g using SP:SAMPLEPREP_SUMMARY an Eppendorf centrifuge 5430 R. The supernatant was transferred into a fresh 1.5 SP:SAMPLEPREP_SUMMARY mL Eppendorf tube and the precipitate was discarded. A 10 µL aliquot of each SP:SAMPLEPREP_SUMMARY sample was pooled to create the pooled biological quality control (PBQC). Ten SP:SAMPLEPREP_SUMMARY µL of each study sample and the PBQC were transferred into HPLC inserts and SP:SAMPLEPREP_SUMMARY evaporated at 30˚C to complete dryness, using a CHRIST RVC 2-33 CD plus speed SP:SAMPLEPREP_SUMMARY vacuum, prior to the GC-MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Shimadzu GC-2010 CH:COLUMN_NAME 30 m Agilent DB-5 column with a diameter of 0.25mm and a film thickness of 1µm CH:SOLVENT_A N/A CH:SOLVENT_B N/A CH:FLOW_GRADIENT N/A CH:FLOW_RATE 1ml/min CH:COLUMN_TEMPERATURE 100 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Shimadzu TQ8050NX MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The GC-MS system used comprised of an AOC6000 autosampler, a 2030 Shimadzu gas MS:MS_COMMENTS chromatograph and a TQ8050NX triple quadrupole mass spectrometer (Shimadzu, MS:MS_COMMENTS Japan)with an electron ionisation source(-70eV). The mass spectrometer was tuned MS:MS_COMMENTS according to the manufacturer’s recommendations using MS:MS_COMMENTS tris-(perfluorobutyl)-amine (CF43). GC-MS was performed on a 30m Agilent DB-5 MS:MS_COMMENTS column with 0.25mm internal diameter column and 1µm film thickness. The MS:MS_COMMENTS injection temperature (inlet) was set at 280°C, the MS transfer line at 280°C MS:MS_COMMENTS and the ion source adjusted to 200°C. Helium was used as the carrier gas at a MS:MS_COMMENTS flow rate of 1 mL/min and argon gas was used in the collision cell to generate MS:MS_COMMENTS the MRM product ion. The analysis of the derivatised samples was performed under MS:MS_COMMENTS the following oven temperature program; 100°C start temperature, hold for 4 MS:MS_COMMENTS minutes, followed by a 10°C/min oven temperature ramp to 320°C with a MS:MS_COMMENTS following final hold for 11 minutes. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ratio of Relative abundance MS_METABOLITE_DATA_START Samples 1-1_Fresh Media 1-2_Fresh Media 1-3_Fresh Media 2-1_HEKWT 2-2_HEKWT 2-3_HEKWT 3-1_HEKWT_A-A 3-2_HEKWT_A-A 3-3_HEKWT_A-A 4-1_HEKSDHAF2_A-A 4-2_HEKSDHAF2_A-A 4-3_HEKSDHAF2_A-A Factors Sample source:1-1_Fresh Media_001 | Treatment:Control neg Sample source:1-1_Fresh Media_002 | Treatment:Control neg Sample source:1-1_Fresh Media_003 | Treatment:Control neg Sample source:2-1_HEKWT_001 | Treatment:Control Sample source:2-1_HEKWT_002 | Treatment:Control Sample source:2-1_HEKWT_003 | Treatment:Control Sample source:3-1_HEKWT_A-A_001 | Treatment:Treatment Sample source:3-1_HEKWT_A-A_002 | Treatment:Treatment Sample source:3-1_HEKWT_A-A_003 | Treatment:Treatment Sample source:4-1_HEKSDHAF2_A-A_001 | Treatment:Treatment Sample source:4-1_HEKSDHAF2_A-A_002 | Treatment:Treatment Sample source:4-1_HEKSDHAF2_A-A_003 | Treatment:Treatment Lactic acid 526142 540035 533392 2214946 1847120 1989117 2325905 2646966 2242141 1686453 1456046 1803902 ISTD_Inositol 2210425 2152864 2271326 2429075 1987070 2166363 2129132 2369576 2165386 2468311 2106197 2515642 Lactic acid/ISTD_Inositol ratio 0.238027529 0.250844921 0.234837271 0.911847514 0.929569668 0.918182687 1.092419352 1.117063137 1.035446336 0.683241698 0.6913152 0.71707421 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB ID Lactic acid HMDB0000190 ISTD_Inositol HMDB0006088 Lactic acid/ISTD_Inositol ratio N/A METABOLITES_END #END