#METABOLOMICS WORKBENCH spinelli_lab_20240501_124346 DATATRACK_ID:4807 STUDY_ID:ST003198 ANALYSIS_ID:AN005248 PROJECT_ID:PR001988 VERSION 1 CREATED_ON May 8, 2024, 8:00 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 mammalian PR:PROJECT_SUMMARY electron carrier that directs electrons to fumarate, instead of O2, as the PR:PROJECT_SUMMARY favored terminal electron acceptor. RQ, which is undetectable in cultured PR:PROJECT_SUMMARY mammalian cells, is enriched in tissues that catalyze fumarate reduction. RQ and PR:PROJECT_SUMMARY UQ-directed ETC circuits support distinct programs of mitochondrial function. PR:PROJECT_SUMMARY Through expression of a bacterial enzyme that converts UQ into RQ and PR:PROJECT_SUMMARY development a novel RQ analog, we demonstrate that reprogramming the mammalian PR:PROJECT_SUMMARY ETC from the UQ to RQ circuit renders cells highly resistant to hypoxia PR:PROJECT_SUMMARY exposure. Thus, we establish RQ as a fundamental component of the mammalian ETC PR:PROJECT_SUMMARY and unveil reprogramming the ETC to the RQ-circuit as a tractable strategy to PR:PROJECT_SUMMARY treat hypoxia-related diseases. PR:INSTITUTE UMass Chan Medical School PR:DEPARTMENT Program in Molecular Medicine PR:LABORATORY Spinelli Lab PR:LAST_NAME Spinelli PR:FIRST_NAME Jessica PR:ADDRESS 55 N Lake Ave, Worcester, MA 01655 PR:EMAIL spinellilab@gmail.com PR:PHONE (508) 856-8989 ext. 68148 #STUDY ST:STUDY_TITLE UQ/RQ panel on human tissue ST:STUDY_SUMMARY The extraction of nonpolar metabolites from the tissues of human for analysis by ST:STUDY_SUMMARY LCMS to measure levels of Ubiquinone and Rhodoquinone. ST:INSTITUTE UMass Chan Medical School ST:DEPARTMENT Program in Molecular Medicine ST:LABORATORY Spinelli Lab ST:LAST_NAME Jerome ST:FIRST_NAME Madison ST:ADDRESS 55 N Lake Ave, Worcester, MA 01655 ST:EMAIL madison.jerome@umassmed.edu ST:PHONE (508) 856-8989 ext. 68148 #SUBJECT SU:SUBJECT_TYPE Human 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 - Mito B01 Sample source:Brain | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_01 SUBJECT_SAMPLE_FACTORS - Mito B02 Sample source:Brain | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_02 SUBJECT_SAMPLE_FACTORS - Mito B03 Sample source:Brain | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_03 SUBJECT_SAMPLE_FACTORS - Mito B04 Sample source:Brain | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_04 SUBJECT_SAMPLE_FACTORS - Mito B05 Sample source:Brain | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_05 SUBJECT_SAMPLE_FACTORS - Mito K01 Sample source:Kidney | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_06 SUBJECT_SAMPLE_FACTORS - Mito K02 Sample source:Kidney | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_07 SUBJECT_SAMPLE_FACTORS - Mito K03 Sample source:Kidney | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_08 SUBJECT_SAMPLE_FACTORS - Mito K04 Sample source:Kidney | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_09 SUBJECT_SAMPLE_FACTORS - Mito K05 Sample source:Kidney | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_10 SUBJECT_SAMPLE_FACTORS - Mito M01 Sample source:Muscle | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_11 SUBJECT_SAMPLE_FACTORS - Mito M02 Sample source:Muscle | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_12 SUBJECT_SAMPLE_FACTORS - Mito M03 Sample source:Muscle | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_13 SUBJECT_SAMPLE_FACTORS - Mito M04 Sample source:Muscle | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_14 SUBJECT_SAMPLE_FACTORS - Mito M05 Sample source:Muscle | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_15 SUBJECT_SAMPLE_FACTORS - Mito AP01 Sample source:Adipose Pannus | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_16 SUBJECT_SAMPLE_FACTORS - Mito AP02 Sample source:Adipose Pannus | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_17 SUBJECT_SAMPLE_FACTORS - Mito AP03 Sample source:Adipose Pannus | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_18 SUBJECT_SAMPLE_FACTORS - Mito AP04 Sample source:Adipose Pannus | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_19 SUBJECT_SAMPLE_FACTORS - Mito AP05 Sample source:Adipose Pannus | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_20 SUBJECT_SAMPLE_FACTORS - WholeTissue B01 Sample source:Brain | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_21 SUBJECT_SAMPLE_FACTORS - WholeTissue B02 Sample source:Brain | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_22 SUBJECT_SAMPLE_FACTORS - WholeTissue B03 Sample source:Brain | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_23 SUBJECT_SAMPLE_FACTORS - WholeTissue B04 Sample source:Brain | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_24 SUBJECT_SAMPLE_FACTORS - WholeTissue B05 Sample source:Brain | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_25 SUBJECT_SAMPLE_FACTORS - WholeTissue K01 Sample source:Kidney | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_26 SUBJECT_SAMPLE_FACTORS - WholeTissue K02 Sample source:Kidney | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_27 SUBJECT_SAMPLE_FACTORS - WholeTissue K03 Sample source:Kidney | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_28 SUBJECT_SAMPLE_FACTORS - WholeTissue K04 Sample source:Kidney | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_29 SUBJECT_SAMPLE_FACTORS - WholeTissue K05 Sample source:Kidney | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_30 SUBJECT_SAMPLE_FACTORS - WholeTissue M01 Sample source:Muscle | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_31 SUBJECT_SAMPLE_FACTORS - WholeTissue M02 Sample source:Muscle | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_32 SUBJECT_SAMPLE_FACTORS - WholeTissue M03 Sample source:Muscle | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_33 SUBJECT_SAMPLE_FACTORS - WholeTissue M04 Sample source:Muscle | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_34 SUBJECT_SAMPLE_FACTORS - WholeTissue M05 Sample source:Muscle | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_35 SUBJECT_SAMPLE_FACTORS - WholeTissue AP01 Sample source:Adipose Pannus | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_36 SUBJECT_SAMPLE_FACTORS - WholeTissue AP02 Sample source:Adipose Pannus | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_37 SUBJECT_SAMPLE_FACTORS - WholeTissue AP03 Sample source:Adipose Pannus | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_38 SUBJECT_SAMPLE_FACTORS - WholeTissue AP04 Sample source:Adipose Pannus | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_39 SUBJECT_SAMPLE_FACTORS - WholeTissue AP05 Sample source:Adipose Pannus | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_40 SUBJECT_SAMPLE_FACTORS - CoQ10 0.001µM Sample source:Standard | Sample type:CoQ10 Standard RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_41 SUBJECT_SAMPLE_FACTORS - CoQ10 0.01µM Sample source:Standard | Sample type:CoQ10 Standard RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_42 SUBJECT_SAMPLE_FACTORS - CoQ10 0.1µM Sample source:Standard | Sample type:CoQ10 Standard RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_43 SUBJECT_SAMPLE_FACTORS - CoQ10 0.25µM Sample source:Standard | Sample type:CoQ10 Standard RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_44 SUBJECT_SAMPLE_FACTORS - CoQ10 0.5µM Sample source:Standard | Sample type:CoQ10 Standard RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_45 SUBJECT_SAMPLE_FACTORS - CoQ10 1µM Sample source:Standard | Sample type:CoQ10 Standard RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_46 SUBJECT_SAMPLE_FACTORS - CoQ10 5µM Sample source:Standard | Sample type:CoQ10 Standard RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_47 SUBJECT_SAMPLE_FACTORS - CoQ10 10µM Sample source:Standard | Sample type:CoQ10 Standard RAW_FILE_NAME(Raw file name)=20240410_MJ46_PV99_Human_48 #COLLECTION CO:COLLECTION_SUMMARY Tissues were harvested, flash frozen in liquid nitrogen, and stored at -80°C CO:COLLECTION_SUMMARY until samples were ready for processing. CO:SAMPLE_TYPE Brain, Kidney, Muscle, & Adipose Pannus #TREATMENT TR:TREATMENT_SUMMARY Untreated #SAMPLEPREP SP:SAMPLEPREP_SUMMARY UQRQ isolation from tissues: Tissues were flash frozen in liquid nitrogen and SP:SAMPLEPREP_SUMMARY powderized using a mortar and pestle. Approximately 10 mg of tissue powder was SP:SAMPLEPREP_SUMMARY transferred into an Eppendorf tube. The tissue powder was re-suspended in 500 SP:SAMPLEPREP_SUMMARY µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl and then vortexed for 15 SP:SAMPLEPREP_SUMMARY minutes at 4°C. 500 µL LCMS-grade hexane (Honeywell) was added to the lysate SP:SAMPLEPREP_SUMMARY and again vortexed for 15 minutes at 4°C. Samples were then centrifuged at SP:SAMPLEPREP_SUMMARY 16,000 x g for 10 minutes at 4°C with the top (hexane) layer containing UQ and SP:SAMPLEPREP_SUMMARY RQ, the middle layer containing the acidified methanol, and the bottom layer SP:SAMPLEPREP_SUMMARY containing the protein. The top layer was transferred into a new Eppendorf tube, SP:SAMPLEPREP_SUMMARY dried down in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to SP:SAMPLEPREP_SUMMARY a CentriVap-105 Cold Trap (Labconco), and stored at -80°C until they were SP:SAMPLEPREP_SUMMARY re-suspended for LC-MS analysis. The acidified methanol layer was discarded, and SP:SAMPLEPREP_SUMMARY the bottom protein layer was saved for protein quantification. The protein layer SP:SAMPLEPREP_SUMMARY from the metabolite isolation was re-suspended in 1 mL of RIPA buffer (150 mM SP:SAMPLEPREP_SUMMARY NaCl, 50 mM Tris HCl pH 7.5, 0.1% SDS, 1% Triton-X 100 (Sigma), 0.5% SP:SAMPLEPREP_SUMMARY deoxycholate (Sigma), cOmplete EDTA-free protease inhibitor (Sigma)). The SP:SAMPLEPREP_SUMMARY samples were vortexed for 10 minutes at 4°C and then centrifuged at 16,000 x g SP:SAMPLEPREP_SUMMARY for 10 minutes at 4°C. Protein concentrations for each sample were calculated SP:SAMPLEPREP_SUMMARY using the Pierce BCA Protein Assay Kit (Life Technologies). The protein SP:SAMPLEPREP_SUMMARY concentrations were used to calculate the re-suspension volume to normalize all SP:SAMPLEPREP_SUMMARY samples for LC-MS analysis to equal (20 µg/µL ) concentration. Isolation of SP:SAMPLEPREP_SUMMARY mitochondria from tissues: Purified mitochondria pellets were subsequently SP:SAMPLEPREP_SUMMARY vortexed in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl for 10 SP:SAMPLEPREP_SUMMARY minutes at 4°C. 500 µL of 100% LCMS-grade hexane (Honeywell) was added to each SP:SAMPLEPREP_SUMMARY tube and vortexed for an additional 10 minutes at 4°C. Samples were then 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. The bottom layer was SP:SAMPLEPREP_SUMMARY discarded and the pellet was saved for protein quantification. Protein was SP:SAMPLEPREP_SUMMARY isolated as described above. Protein concentrations were calculated using the SP:SAMPLEPREP_SUMMARY Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations SP:SAMPLEPREP_SUMMARY were used to calculate the re-suspension volume to normalize all samples for SP:SAMPLEPREP_SUMMARY LC-MS analysis to equal (20 µg/uL) concentration. Isolation of UQ/RQ from SP:SAMPLEPREP_SUMMARY purified mitochondria: Purified mitochondria pellets were subsequently vortexed SP:SAMPLEPREP_SUMMARY in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl for 10 minutes at SP:SAMPLEPREP_SUMMARY 4°C. 500 µL of 100% LCMS-grade hexane (Honeywell) was added to each tube and SP:SAMPLEPREP_SUMMARY vortexed for an additional 10 minutes at 4°C. Samples were then centrifuged for SP:SAMPLEPREP_SUMMARY 10 minutes at 21,300 x g at 4°C. The top (hexane) layer of the sample SP:SAMPLEPREP_SUMMARY (containing UQ and RQ) was transferred to a new labeled 1.5 mL Eppendorf tube SP:SAMPLEPREP_SUMMARY and dried in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to SP:SAMPLEPREP_SUMMARY a CentriVap-105 Cold Trap (Labconco). Samples were stored in -80°C freezer SP:SAMPLEPREP_SUMMARY prior to resuspension and running on LC-MS. The bottom layer was discarded and SP:SAMPLEPREP_SUMMARY the pellet was saved for protein quantification. Protein was isolated as SP:SAMPLEPREP_SUMMARY described above. Protein concentrations were calculated using the Pierce BCA SP:SAMPLEPREP_SUMMARY Protein Assay Kit (Life Technologies). The protein concentrations were used to SP:SAMPLEPREP_SUMMARY calculate the re-suspension volume to normalize all samples for LC-MS analysis SP:SAMPLEPREP_SUMMARY to equal (20 µg/uL) concentration. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Luna PFP(2) (100 x 2mm,3µm) 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. MS:MS_RESULTS_FILE ST003198_AN005248_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END