#METABOLOMICS WORKBENCH spinelli_lab_20240520_111432 DATATRACK_ID:4850 STUDY_ID:ST003218 ANALYSIS_ID:AN005277 PROJECT_ID:PR001988 VERSION 1 CREATED_ON May 22, 2024, 7:58 pm #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 Glutamine Tracing Assay in WT and SDHB KO 143B cells Treated with HKJS003 ST:STUDY_SUMMARY WT and succinate dehydrogenase iron sulfur subunit B(SDHB) KO 143B cells were ST:STUDY_SUMMARY treated with a small molecule analog of Rhodoquinone, HKJS003. Glutamine tracing ST:STUDY_SUMMARY was used to determine the fumarate reduction and succinate oxidation of the ST:STUDY_SUMMARY treated cells. Other metabolites such as ATP,ADP,AMP,Glutathione, Oxidized ST:STUDY_SUMMARY glutathione, UTP, lactic acid, and pyruvic acid were also measured. 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 #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 - WT 1 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_01_20240402125032.raw SUBJECT_SAMPLE_FACTORS - WT 2 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_02.raw SUBJECT_SAMPLE_FACTORS - WT 3 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_03.raw SUBJECT_SAMPLE_FACTORS - WT 25nM 003 1 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_04.raw SUBJECT_SAMPLE_FACTORS - WT 25nM 003 2 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_05.raw SUBJECT_SAMPLE_FACTORS - WT 25nM 003 3 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_06.raw SUBJECT_SAMPLE_FACTORS - WT 100nM 003 1 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_07.raw SUBJECT_SAMPLE_FACTORS - WT 100nM 003 2 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_08.raw SUBJECT_SAMPLE_FACTORS - WT 100nM 003 3 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_09.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 1 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_10.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 2 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_11.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 3 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_12.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 25nM 003 1 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_13.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 25nM 003 2 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_14.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 25nM 003 3 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_15.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 100nM 003 1 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_16.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 100nM 003 2 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_17.raw SUBJECT_SAMPLE_FACTORS - SDHB KO 100nM 003 3 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003 Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_18.raw #COLLECTION CO:COLLECTION_SUMMARY Media was aspirated from the plates and then the cells were washed with 1x PBS CO:COLLECTION_SUMMARY twice. The plate was then transferred to dry ice and 500 µL of 80% HPLC-grade CO:COLLECTION_SUMMARY methanol (Sigma) 20% HPLC-grade water (Sigma) was added to each well. The wells CO:COLLECTION_SUMMARY were placed in a -80 freezer to incubate for 15 minutes. The plates are taken CO:COLLECTION_SUMMARY out of the freezer one at a time and placed back on dry ice. The cells were then CO:COLLECTION_SUMMARY scraped and transferred to a new 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.Cells were then supplemented with either DMSO or TR:TREATMENT_SUMMARY HKJS003. For HKJS003, 20µL or 5µL of 100µM stock was added to 2mL of media TR:TREATMENT_SUMMARY for a final concentration of 100nM or 25nM. For DMSO, 20µL of a diluted 1:1000 TR:TREATMENT_SUMMARY DMSO was added to 2mL of media. The cells were incubated for 5-7 of days to TR:TREATMENT_SUMMARY proliferate and every 2 days the media was refreshed with the relevant TR:TREATMENT_SUMMARY treatments. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Once cells were collected into Eppendorf tubes, each well was washed with an SP:SAMPLEPREP_SUMMARY additional 300 µL of 80% HPLC-grade methanol (Sigma) 20% HPLC-grade water SP:SAMPLEPREP_SUMMARY (Sigma) and collected into the same tube as the initial lysis. The samples were SP:SAMPLEPREP_SUMMARY then vortexed at 4° C for 10 minutes and centrifuged at 21,300 x g for 10 SP:SAMPLEPREP_SUMMARY minutes at 4° C. Supernatants were transferred to a new tube and dried down in SP:SAMPLEPREP_SUMMARY a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a SP:SAMPLEPREP_SUMMARY CentriVap-105 Cold Trap (Labconco). After being dried down, pellets were stored SP:SAMPLEPREP_SUMMARY in a -20° C freezer until ready to run on the polar LC-MS method. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 20 min, 80% to 20% B; 0.5 min, 20% to 80% B; 7.5min, 80% B CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) CH:SOLVENT_A 100% water; 0.1% ammonium hydroxide; 20mM ammonium carbonate CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT Gradient starts at 80% B. Subsequently, 80% to 20% B linearly in 20min; 20% to CH:FLOW_GRADIENT 80% B in 0.5min; 80% B for 7.5min CH:FLOW_RATE 0.15ml/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 NEGATIVE MS:MS_COMMENTS The mass spectrometer had the spray voltage set to 4.0 kV, heated capillary to MS:MS_COMMENTS 350°C, and the HESI probe at 30 °C. The sheath gas flow was set at 10 units, MS:MS_COMMENTS auxiliary gas at 1 units, and sweep gas flow at 1 unit. An additional scan MS:MS_COMMENTS between 220-700 m/z was used to enhance nucleotide detection in the negative MS:MS_COMMENTS mode as well with the maximum injection time set to 80 msec. Data acquired by MS:MS_COMMENTS Thermo Fisher's Xcalibur software and analyzed by their Tracefinder software. MS:MS_COMMENTS The raw files provided contain data from both positive and negative ion mode. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples WT 1 WT 2 WT 3 WT 25nM 003 1 WT 25nM 003 2 WT 25nM 003 3 WT 100nM 003 1 WT 100nM 003 2 WT 100nM 003 3 SDHB KO 1 SDHB KO 2 SDHB KO 3 SDHB KO 25nM 003 1 SDHB KO 25nM 003 2 SDHB KO 25nM 003 3 SDHB KO 100nM 003 1 SDHB KO 100nM 003 2 SDHB KO 100nM 003 3 Factors Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003 Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003 Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003 Fumaric acid 28506723.02 32588514.68 32380756.52 35151508.77 35892243.27 28645875.37 20808601.95 26892502.9 24148779.43 31060426.77 31104001.7 33237898.69 33441144.26 34448381.59 41237284.97 20429099.55 31845333.13 30444344.37 Fumaric acid_13C1 3649573.465 4077450.956 4008654.557 3754703.601 3951219.286 3182456.633 1025133.619 1496108.198 1137069.185 3760143.177 3957764.756 4214336.818 3755564.249 3772378.261 4698464.56 932263.4396 1576589.08 1462482.649 Fumaric acid_13C2 7428137.855 7967973.405 8372138.322 7886440.667 8210696.999 6705069.009 2070073.749 2606087.415 2054281.589 7938783.503 8307697.67 8889547.597 8650125.828 8772026.049 10705450.93 1771175.958 2619658.945 2428414.241 Fumaric acid_13C3 8125881.37 8091406.287 8279617.838 10904585.49 10943270.77 9084147.95 14170343.61 17705021.74 16817582.79 7342897.854 7204089.703 8231143.221 9534777.822 9337250.563 11455172.87 13475641.44 20603036.55 19372224.28 Fumaric acid_13C4 28093676.98 28810615.69 30093849.58 30724350.75 31479625.14 25340498.94 11711257.8 14854140.59 12465117.68 33334446.79 34848668.06 38144564.83 34668827.88 36536579.15 44363956.1 10804058.27 15320116.53 14713273.97 Succinic acid 4784588.765 3446494.472 4796852.824 3781966.297 3283601.389 2459375.722 5753541.484 9414509.622 7927386.285 8141343.868 5764541.22 8795082.948 8386425.718 8844122.26 10656253.33 13871288.09 23568791.53 25512927.31 Succinic acid_13C1 635769.6608 575013.1944 597028.7253 684276.6795 529149.7108 433437.3801 461379.489 578933.7647 617739.8631 1159193.402 1012177.696 1271246.148 1250790.771 1303807.727 1831928.493 820671.8107 1436421.557 1697039.243 Succinic acid_13C2 426806.7799 565082.0641 536310.8916 338499.7152 513331.4359 337919.6586 517199.8373 783625.8469 240612.6716 1538885.146 1227151.867 2054960.335 2132330.248 2251606.981 3119600.399 1310343.893 3695986.162 3367546.748 Succinic acid_13C3 142776.7064 45588.67683 192174.1166 244289.0767 341050.475 208935.991 705817.5845 884311.7645 1058991.18 740242.6699 575792.9481 786306.7626 727574.9975 789641.1068 1053402.219 1826538.391 3428133.385 3772581.566 Succinic acid_13C4 4626503.85 4073921.413 5940112.438 7659099.867 5959272.531 5437855.37 17096278.04 25412649.09 24783371.18 16071039.15 15451783.72 18887161.8 18940232.7 20562312.43 27699202.98 46740962.52 87609447.44 90249984.63 UTP 29083136.22 33606136.54 31691640.31 26660552.27 21008994.69 14352760.47 26197831.71 44599582.6 45613862.71 48681242.52 47437423.59 52799628.67 32451183.17 30211692.5 43539044.31 36008773.84 60619474.6 66534792.75 ATP 56248428.08 66885455.53 62748967.99 49436107.05 44008855.8 28929788.08 45142541.57 79553925.18 73553858.65 77729211.29 75309399.99 85405378.71 59084521.54 54240852.47 78257948.94 62938603.2 95119636.05 109589752.2 AMP 4912132.048 5007798.019 4652157.822 5231344.798 4709067.41 3164420.733 4601706.176 7113253.756 7239005.794 4764396.325 4603569.818 4300497.084 4682551.655 4634371.615 5858806.183 4472695.81 6680887.406 6137765.624 ADP 8765303.192 10150914.2 8911014.028 8726956.354 8086551.75 5380049.926 7482025.129 10130335.68 9596911.941 9065397.635 7873364.513 6823904.223 6496916.17 6948526.468 8125020.676 8543298.739 12269469.08 11865971.53 Lactic acid 1522264718 1167188947 1442277568 1194019068 1077316487 709306346.1 1035515031 1413307717 1363584154 1173161149 819096113.5 1107970572 1121912981 924312057.7 1236821726 1427046411 1986029214 2035345635 Pyruvic acid 13910843.7 10400047.36 14197461.67 10835639.22 7547966.269 6892407.765 12215953.24 12608946.58 8811691.678 14494231.15 8390943.347 10346654.32 10702867.85 8205012.961 11170269.43 13475805 13076794.91 10331109.64 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention index Quantified m/z PubChem ID KEGG ID Fumaric acid 10.15 115.00368 444972 C00122 Fumaric acid_13C1 10.15 116.00704 Fumaric acid_13C2 10.15 117.01039 Fumaric acid_13C3 10.15 118.01375 Fumaric acid_13C4 10.15 119.0171 Succinic acid 9.62 117.01933 1110 C00042 Succinic acid_13C1 9.62 118.02269 Succinic acid_13C2 9.62 119.02604 Succinic acid_13C3 9.62 120.0294 Succinic acid_13C4 9.62 121.03275 UTP 12.02 482.96125 6133 C00075 ATP 10.77 505.98847 5957 C00002 AMP 8.51 346.05581 6083 C00020 ADP 9.59 426.02214 6022 C00008 Lactic acid 5.36 89.02442 107689 C00186 Pyruvic acid 4.34 87.00877 1060 C00022 METABOLITES_END #END