{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003198","ANALYSIS_ID":"AN005248","PROJECT_ID":"PR001988","VERSION":"1","CREATED_ON":"May 8, 2024, 8:00 am"},

"PROJECT":{"PROJECT_TITLE":"Rhodoquinone is an Electron Carrier in the Mammalian Electron Transport Chain","PROJECT_SUMMARY":"Ubiquinone (UQ), the only known electron carrier in the mammalian electron transport chain (ETC), delivers electrons to both oxygen (O2) and fumarate as terminal electron acceptors. As fumarate has a lower reduction potential than UQ, fumarate reduction is only thermodynamically favorable when ubiquinol, the reduced form of UQ, accumulates. Paradoxically, some tissues reduce fumarate without ubiquinol buildup, suggesting another mechanism enables fumarate reduction in mammals. Here, we identify rhodoquinone (RQ), a novel mammalian electron carrier that directs electrons to fumarate, instead of O2, as the favored terminal electron acceptor. RQ, which is undetectable in cultured mammalian cells, is enriched in tissues that catalyze fumarate reduction. RQ and UQ-directed ETC circuits support distinct programs of mitochondrial function. Through expression of a bacterial enzyme that converts UQ into RQ and development a novel RQ analog, we demonstrate that reprogramming the mammalian ETC from the UQ to RQ circuit renders cells highly resistant to hypoxia exposure. Thus, we establish RQ as a fundamental component of the mammalian ETC and unveil reprogramming the ETC to the RQ-circuit as a tractable strategy to treat hypoxia-related diseases.","INSTITUTE":"UMass Chan Medical School","DEPARTMENT":"Program in Molecular Medicine","LABORATORY":"Spinelli Lab","LAST_NAME":"Spinelli","FIRST_NAME":"Jessica","ADDRESS":"55 N Lake Ave, Worcester, MA 01655","EMAIL":"spinellilab@gmail.com","PHONE":"(508) 856-8989 ext. 68148"},

"STUDY":{"STUDY_TITLE":"UQ/RQ panel on human tissue","STUDY_SUMMARY":"The extraction of nonpolar metabolites from the tissues of human for analysis by LCMS to measure levels of Ubiquinone and Rhodoquinone.","INSTITUTE":"UMass Chan Medical School","DEPARTMENT":"Program in Molecular Medicine","LABORATORY":"Spinelli Lab","LAST_NAME":"Jerome","FIRST_NAME":"Madison","ADDRESS":"55 N Lake Ave, Worcester, MA 01655","EMAIL":"madison.jerome@umassmed.edu","PHONE":"(508) 856-8989 ext. 68148"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Mito B01",
"Factors":{"Sample source":"Brain","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_01"}
},
{
"Subject ID":"-",
"Sample ID":"Mito B02",
"Factors":{"Sample source":"Brain","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_02"}
},
{
"Subject ID":"-",
"Sample ID":"Mito B03",
"Factors":{"Sample source":"Brain","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_03"}
},
{
"Subject ID":"-",
"Sample ID":"Mito B04",
"Factors":{"Sample source":"Brain","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_04"}
},
{
"Subject ID":"-",
"Sample ID":"Mito B05",
"Factors":{"Sample source":"Brain","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_05"}
},
{
"Subject ID":"-",
"Sample ID":"Mito K01",
"Factors":{"Sample source":"Kidney","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_06"}
},
{
"Subject ID":"-",
"Sample ID":"Mito K02",
"Factors":{"Sample source":"Kidney","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_07"}
},
{
"Subject ID":"-",
"Sample ID":"Mito K03",
"Factors":{"Sample source":"Kidney","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_08"}
},
{
"Subject ID":"-",
"Sample ID":"Mito K04",
"Factors":{"Sample source":"Kidney","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_09"}
},
{
"Subject ID":"-",
"Sample ID":"Mito K05",
"Factors":{"Sample source":"Kidney","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_10"}
},
{
"Subject ID":"-",
"Sample ID":"Mito M01",
"Factors":{"Sample source":"Muscle","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_11"}
},
{
"Subject ID":"-",
"Sample ID":"Mito M02",
"Factors":{"Sample source":"Muscle","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_12"}
},
{
"Subject ID":"-",
"Sample ID":"Mito M03",
"Factors":{"Sample source":"Muscle","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_13"}
},
{
"Subject ID":"-",
"Sample ID":"Mito M04",
"Factors":{"Sample source":"Muscle","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_14"}
},
{
"Subject ID":"-",
"Sample ID":"Mito M05",
"Factors":{"Sample source":"Muscle","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_15"}
},
{
"Subject ID":"-",
"Sample ID":"Mito AP01",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_16"}
},
{
"Subject ID":"-",
"Sample ID":"Mito AP02",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_17"}
},
{
"Subject ID":"-",
"Sample ID":"Mito AP03",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_18"}
},
{
"Subject ID":"-",
"Sample ID":"Mito AP04",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_19"}
},
{
"Subject ID":"-",
"Sample ID":"Mito AP05",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Mitochondria"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_20"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue B01",
"Factors":{"Sample source":"Brain","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_21"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue B02",
"Factors":{"Sample source":"Brain","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_22"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue B03",
"Factors":{"Sample source":"Brain","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_23"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue B04",
"Factors":{"Sample source":"Brain","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_24"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue B05",
"Factors":{"Sample source":"Brain","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_25"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue K01",
"Factors":{"Sample source":"Kidney","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_26"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue K02",
"Factors":{"Sample source":"Kidney","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_27"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue K03",
"Factors":{"Sample source":"Kidney","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_28"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue K04",
"Factors":{"Sample source":"Kidney","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_29"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue K05",
"Factors":{"Sample source":"Kidney","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_30"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue M01",
"Factors":{"Sample source":"Muscle","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_31"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue M02",
"Factors":{"Sample source":"Muscle","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_32"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue M03",
"Factors":{"Sample source":"Muscle","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_33"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue M04",
"Factors":{"Sample source":"Muscle","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_34"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue M05",
"Factors":{"Sample source":"Muscle","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_35"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue AP01",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_36"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue AP02",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_37"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue AP03",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_38"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue AP04",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_39"}
},
{
"Subject ID":"-",
"Sample ID":"WholeTissue AP05",
"Factors":{"Sample source":"Adipose Pannus","Sample type":"Whole Tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_40"}
},
{
"Subject ID":"-",
"Sample ID":"CoQ10 0.001µM",
"Factors":{"Sample source":"Standard","Sample type":"CoQ10 Standard"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_41"}
},
{
"Subject ID":"-",
"Sample ID":"CoQ10 0.01µM",
"Factors":{"Sample source":"Standard","Sample type":"CoQ10 Standard"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_42"}
},
{
"Subject ID":"-",
"Sample ID":"CoQ10 0.1µM",
"Factors":{"Sample source":"Standard","Sample type":"CoQ10 Standard"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_43"}
},
{
"Subject ID":"-",
"Sample ID":"CoQ10 0.25µM",
"Factors":{"Sample source":"Standard","Sample type":"CoQ10 Standard"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_44"}
},
{
"Subject ID":"-",
"Sample ID":"CoQ10 0.5µM",
"Factors":{"Sample source":"Standard","Sample type":"CoQ10 Standard"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_45"}
},
{
"Subject ID":"-",
"Sample ID":"CoQ10 1µM",
"Factors":{"Sample source":"Standard","Sample type":"CoQ10 Standard"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_46"}
},
{
"Subject ID":"-",
"Sample ID":"CoQ10 5µM",
"Factors":{"Sample source":"Standard","Sample type":"CoQ10 Standard"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_47"}
},
{
"Subject ID":"-",
"Sample ID":"CoQ10 10µM",
"Factors":{"Sample source":"Standard","Sample type":"CoQ10 Standard"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"20240410_MJ46_PV99_Human_48"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Tissues were harvested, flash frozen in liquid nitrogen, and stored at -80°C until samples were ready for processing.","SAMPLE_TYPE":"Brain, Kidney, Muscle, & Adipose Pannus"},

"TREATMENT":{"TREATMENT_SUMMARY":"Untreated"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"UQRQ isolation from tissues: Tissues were flash frozen in liquid nitrogen and powderized using a mortar and pestle. Approximately 10 mg of tissue powder was transferred into an Eppendorf tube. The tissue powder was re-suspended in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl and then vortexed for 15 minutes at 4°C. 500 µL LCMS-grade hexane (Honeywell) was added to the lysate and again vortexed for 15 minutes at 4°C. Samples were then centrifuged at 16,000 x g for 10 minutes at 4°C with the top (hexane) layer containing UQ and RQ, the middle layer containing the acidified methanol, and the bottom layer containing the protein. The top layer was transferred into a new Eppendorf tube, dried down in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco), and stored at -80°C until they were re-suspended for LC-MS analysis. The acidified methanol layer was discarded, and the bottom protein layer was saved for protein quantification. The protein layer from the metabolite isolation was re-suspended in 1 mL of RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 0.1% SDS, 1% Triton-X 100 (Sigma), 0.5% deoxycholate (Sigma), cOmplete EDTA-free protease inhibitor (Sigma)). The samples were vortexed for 10 minutes at 4°C and then centrifuged at 16,000 x g for 10 minutes at 4°C. Protein concentrations for each sample were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/µL ) concentration. Isolation of mitochondria from tissues: Purified mitochondria pellets were subsequently vortexed in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl for 10 minutes at 4°C. 500 µL of 100% LCMS-grade hexane (Honeywell) was added to each tube and vortexed for an additional 10 minutes at 4°C. Samples were then centrifuged for 10 minutes at 21,300 x g at 4°C. The top (hexane) layer of the sample (containing UQ and RQ) was transferred to a new labeled 1.5 mL Eppendorf tube and dried in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). Samples were stored in -80°C freezer prior to resuspension and running on LC-MS. The bottom layer was discarded and the pellet was saved for protein quantification. Protein was isolated as described above. Protein concentrations were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/uL) concentration. Isolation of UQ/RQ from purified mitochondria: Purified mitochondria pellets were subsequently vortexed in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl for 10 minutes at 4°C. 500 µL of 100% LCMS-grade hexane (Honeywell) was added to each tube and vortexed for an additional 10 minutes at 4°C. Samples were then centrifuged for 10 minutes at 21,300 x g at 4°C. The top (hexane) layer of the sample (containing UQ and RQ) was transferred to a new labeled 1.5 mL Eppendorf tube and dried in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). Samples were stored in -80°C freezer prior to resuspension and running on LC-MS. The bottom layer was discarded and the pellet was saved for protein quantification. Protein was isolated as described above. Protein concentrations were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/uL) concentration."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Luna PFP(2) (100 x 2mm,3µm)","SOLVENT_A":"100% water; 0.1% formic acid","SOLVENT_B":"100% acetonitrile; 0.1% formic acid","FLOW_GRADIENT":"0 - 3 min, 30% A; 3 - 3.25 min, 30% - 2% A; 3.25 - 5min, 2% A; 5 - 6min, 2% - 1% A; 6 - 8.75min, 1% A; 8.75 - 9min, 1% - 30% A; 9 - 10min, 30% A","FLOW_RATE":"0.5mL/min","COLUMN_TEMPERATURE":"25"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The mass spectrometer was operated in full scan, positive-ion mode, with the spray voltage set to 3.0 kV, the heated capillary at 275°C, and the HESI probe at 350°C. The sheath gas flow was 40 units, the auxiliary gas flow was 15 units, and the sweep gas flow was 1 unit. MS data was collected in a range of m/z = 200 –1000. The resolution set at 17,500, the AGC target at 3x106, and the maximum injection time at 250 msec. Data acquired by Thermo Fisher''s Xcalibur software and analyzed by their Tracefinder software.","MS_RESULTS_FILE":"ST003198_AN005248_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}