#METABOLOMICS WORKBENCH spinelli_lab_20240520_072814 DATATRACK_ID:4849 STUDY_ID:ST003217 ANALYSIS_ID:AN005275 PROJECT_ID:PR001988 VERSION 1 CREATED_ON May 22, 2024, 9:36 am #PROJECT PR:PROJECT_TITLE Rhodoquinone is an Electron Carrier in the Mammalian Electron Transport Chain PR:PROJECT_SUMMARY Ubiquinone (UQ), the only known electron carrier in the mammalian electron PR:PROJECT_SUMMARY transport chain (ETC), delivers electrons to both oxygen (O2) and fumarate as PR:PROJECT_SUMMARY terminal electron acceptors. As fumarate has a lower reduction potential than PR:PROJECT_SUMMARY UQ, fumarate reduction is only thermodynamically favorable when ubiquinol, the PR:PROJECT_SUMMARY reduced form of UQ, accumulates. Paradoxically, some tissues reduce fumarate PR:PROJECT_SUMMARY without ubiquinol buildup, suggesting another mechanism enables fumarate PR:PROJECT_SUMMARY reduction in mammals. Here, we identify rhodoquinone (RQ), a novel component of PR:PROJECT_SUMMARY the mammalian ETC that carries electrons to fumarate, instead of O2, as the PR:PROJECT_SUMMARY terminal electron acceptor. RQ, which is undetectable in cultured mammalian PR:PROJECT_SUMMARY cells, is enriched in mitochondria from mouse and human tissues that catalyze PR:PROJECT_SUMMARY high levels of fumarate reduction. UQ and RQ-directed ETC circuits support PR:PROJECT_SUMMARY unique programs of mitochondrial function. Through expression of a bacterial PR:PROJECT_SUMMARY enzyme that converts UQ into RQ and development of a novel RQ analog, we PR:PROJECT_SUMMARY demonstrate that reprogramming the mammalian ETC from the UQ to RQ circuit PR:PROJECT_SUMMARY renders cells highly resistant to hypoxia exposure in vitro and in vivo. Thus, PR:PROJECT_SUMMARY we establish RQ as a fundamental component of the mammalian ETC and unveil that PR:PROJECT_SUMMARY reprogramming the ETC to the RQ-circuit is a tractable strategy to ameliorate PR:PROJECT_SUMMARY hypoxia-related conditions. PR:INSTITUTE UMass Chan Medical School PR:LAST_NAME UMass Chan PR:FIRST_NAME Spinelli Lab PR:ADDRESS 55 Lake Avenue North, Worcester, Massachusetts, 01605, USA PR:EMAIL spinellilab@gmail.com PR:PHONE (508) 856-8989 ext. 68148 #STUDY ST:STUDY_TITLE Measuring UQ/UQH2 in WT and RquA 143B cell in Hypoxia ST:STUDY_SUMMARY Cultured cells were used to measure the ratio of UQ to UQH2 in WT and RquA 143B ST:STUDY_SUMMARY cells. Cells were exposed to hypoxia to see if there was a difference in UQ:UQH2 ST:STUDY_SUMMARY ratio. ST:INSTITUTE UMass Chan Medical School ST:LAST_NAME UMass Chan ST:FIRST_NAME Spinelli Lab ST:ADDRESS 55 Lake Avenue North, Worcester, Massachusetts, 01605, USA ST:EMAIL spinellilab@gmail.com ST:PHONE (508) 856-8989 ext. 68148 #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 - WT 1 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:normoxia Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_01_20240322180453.raw SUBJECT_SAMPLE_FACTORS - WT 2 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:normoxia Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_02_20240322181535.raw SUBJECT_SAMPLE_FACTORS - WT 3 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:normoxia Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_03_20240322182615.raw SUBJECT_SAMPLE_FACTORS - WT Hypoxia 1 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:hypoxia exposed Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_04_20240322183655.raw SUBJECT_SAMPLE_FACTORS - WT Hypoxia 2 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:hypoxia exposed Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_05_20240322184736.raw SUBJECT_SAMPLE_FACTORS - WT Hypoxia 3 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:hypoxia exposed Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_06_20240322185815.raw SUBJECT_SAMPLE_FACTORS - RquA 1 Sample source:Bone sarcoma cells | Genotype:RquA expressing | Treatment:normoxia Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_07_20240322190855.raw SUBJECT_SAMPLE_FACTORS - RquA 2 Sample source:Bone sarcoma cells | Genotype:RquA expressing | Treatment:normoxia Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_08_20240322191935.raw SUBJECT_SAMPLE_FACTORS - RquA 3 Sample source:Bone sarcoma cells | Genotype:RquA expressing | Treatment:normoxia Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_09_20240322193016.raw SUBJECT_SAMPLE_FACTORS - RquA Hypoxia 1 Sample source:Bone sarcoma cells | Genotype:RquA expressing | Treatment:hypoxia exposed Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_10_20240322194055.raw SUBJECT_SAMPLE_FACTORS - RquA Hypoxia 2 Sample source:Bone sarcoma cells | Genotype:RquA expressing | Treatment:hypoxia exposed Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_11_20240322195136.raw SUBJECT_SAMPLE_FACTORS - RquA Hypoxia 3 Sample source:Bone sarcoma cells | Genotype:RquA expressing | Treatment:hypoxia exposed Cell line=143B; RAW_FILE_NAME(File Name )=20240311_JV_UQRQ_NVH_12_20240322200215.raw #COLLECTION CO:COLLECTION_SUMMARY Plates were removed from incubator, media was aspirated, wells were washed with CO:COLLECTION_SUMMARY 1X PBS, and the wash was aspirated. Plates were moved to dry ice and 500 µL of CO:COLLECTION_SUMMARY 100% LCMS-grade ethanol (Sigma) was added to each well. Cells were then scraped CO:COLLECTION_SUMMARY with cell scraper and lysate was moved to a labeled and pre-cooled 1.5 mL CO:COLLECTION_SUMMARY Eppendorf tube. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY The 143B cell line were cultured in Dulbecco's Modified Eagle Medium (DMEM) TR:TREATMENT_SUMMARY (ThermoFisher) supplemented with 10% Heat Inactivated Fetal Bovine Serum TR:TREATMENT_SUMMARY (ThermoFisher) and 1% penicillin and streptomycin (ThermoFisher), and 100 µg/mL TR:TREATMENT_SUMMARY uridine (Sigma). Cells were kept in a 37°C incubator (Baker Ruskinn) held at 5% TR:TREATMENT_SUMMARY CO2 and 90% relative humidity. 25,000 – 50,000 cells were seeded in complete TR:TREATMENT_SUMMARY DMEM media. 24 hours later the media was changed with 1.25µL of 10mg/mL of TR:TREATMENT_SUMMARY doxycycline in 50mL of DMEM to induce RquA expression. The cells were incubated TR:TREATMENT_SUMMARY for 5-7 of days to proliferate and every 2 days the media was refreshed. For TR:TREATMENT_SUMMARY conditions done in hypoxia, cells were initially seeded in normoxia. 24 hours TR:TREATMENT_SUMMARY after seeding, the cells are transferred into the Invivo2 1000 workstation TR:TREATMENT_SUMMARY (Baker Ruskinn) with the oxygen tension set to 0.5% O2, the temperature to TR:TREATMENT_SUMMARY 37°C, the CO2 to 5%, and the relative humidity to 80%. Media would be changed TR:TREATMENT_SUMMARY in the Invivo2 1000 workstation the same as normoxia. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Once cells were collected into Eppendorf tubes,samples were then vortexed for 10 SP:SAMPLEPREP_SUMMARY minutes in a cold room. Then, 1 mL of 100% LCMS-grade hexane (sigma) added to it SP:SAMPLEPREP_SUMMARY and samples were vortexed for another 10 minutes in the cold room. Samples were SP:SAMPLEPREP_SUMMARY centrifuged for 10 minutes at 21,300 x g at 4°C. The top (hexane) layer of the SP:SAMPLEPREP_SUMMARY sample (containing UQ and RQ) was transferred to a new labeled 1.5 mL Eppendorf SP:SAMPLEPREP_SUMMARY tube and dried in a Refrigerated CentriVap Benchtop Vacuum Concentrator SP:SAMPLEPREP_SUMMARY connected to a CentriVap-105 Cold Trap (Labconco). Samples were stored in -80°C SP:SAMPLEPREP_SUMMARY freezer prior to resuspension and running on LC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Luna PFP(2) (100 x 2mm, 3um)) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0 - 3 min, 30% A; 3 - 3.25 min, 30% - 2% A; 3.25 - 5min, 2% A; 5 - 6min, 2% - 1% CH:FLOW_GRADIENT A; 6 - 8.75min, 1% A; 8.75 - 9min, 1% - 30% A; 9 - 10min, 30% A CH:FLOW_RATE 0.5mL/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometer was operated in full scan, positive-ion mode, with the MS:MS_COMMENTS spray voltage set to 3.0 kV, the heated capillary at 275°C, and the HESI probe MS:MS_COMMENTS at 350°C. The sheath gas flow was 40 units, the auxiliary gas flow was 15 MS:MS_COMMENTS units, and the sweep gas flow was 1 unit. MS data was collected in a range of MS:MS_COMMENTS m/z = 200 –1000. The resolution set at 17,500, the AGC target at 3x106, and MS:MS_COMMENTS the maximum injection time at 250 msec. Data acquired by Thermo Fisher's MS:MS_COMMENTS Xcalibur software and analyzed by their Tracefinder software. The data reported MS:MS_COMMENTS includes the different adducts and reduced forms of the metabolites to show MS:MS_COMMENTS presence or lack thereof. MS:MS_RESULTS_FILE ST003217_AN005275_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END