#METABOLOMICS WORKBENCH spinelli_lab_20240725_130516 DATATRACK_ID:5034 STUDY_ID:ST003397 ANALYSIS_ID:AN005575 PROJECT_ID:PR001988 VERSION 1 CREATED_ON August 12, 2024, 11:39 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 Glutamine Tracing in Mice injected with HKJS001 and Malonic Acid ST:STUDY_SUMMARY Mice were injected by IP with 13C515N2-glutamine solution. Each mice was ST:STUDY_SUMMARY injected with 15uL of 50mg/mL glutamine solution per gram of mouse. Mice were ST:STUDY_SUMMARY injected with DMSO as a control, HKJS001 compound, 1M malonic acid, or a ST:STUDY_SUMMARY combination of HKJS001 and Malonic Acid. The mice were sacrificed and the liver ST:STUDY_SUMMARY was harvested. Polar metabolites extraction performed on the liver tissue. 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 Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - Liver 1 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Vehicle RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_01 SUBJECT_SAMPLE_FACTORS - Liver 2 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Vehicle RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_02 SUBJECT_SAMPLE_FACTORS - Liver 3 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Vehicle RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_03 SUBJECT_SAMPLE_FACTORS - Liver 4 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Vehicle RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_04 SUBJECT_SAMPLE_FACTORS - Liver 5 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:HKJS001 Injected RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_05 SUBJECT_SAMPLE_FACTORS - Liver 6 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:HKJS001 Injected RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_06 SUBJECT_SAMPLE_FACTORS - Liver 7 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:HKJS001 Injected RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_07 SUBJECT_SAMPLE_FACTORS - Liver 8 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:HKJS001 Injected RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_08 SUBJECT_SAMPLE_FACTORS - Liver 9 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Malonic Acid Injected RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_09 SUBJECT_SAMPLE_FACTORS - Liver 10 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Malonic Acid Injected RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_10 SUBJECT_SAMPLE_FACTORS - Liver 11 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Malonic Acid Injected RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_11 SUBJECT_SAMPLE_FACTORS - Liver 12 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Malonic Acid Injected RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_12 SUBJECT_SAMPLE_FACTORS - Liver 13 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Combo HKJS001 and Malonic Acid RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_13 SUBJECT_SAMPLE_FACTORS - Liver 14 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Combo HKJS001 and Malonic Acid RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_14 SUBJECT_SAMPLE_FACTORS - Liver 15 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Combo HKJS001 and Malonic Acid RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_15 SUBJECT_SAMPLE_FACTORS - Liver 16 Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Combo HKJS001 and Malonic Acid RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_16 SUBJECT_SAMPLE_FACTORS - Standard 2 Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_17 SUBJECT_SAMPLE_FACTORS - Standard 3 Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_18 SUBJECT_SAMPLE_FACTORS - Standard 4 Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_19 SUBJECT_SAMPLE_FACTORS - Standard 5 Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_20 SUBJECT_SAMPLE_FACTORS - Standard 6 Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_21 SUBJECT_SAMPLE_FACTORS - Standard 7 Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_22 SUBJECT_SAMPLE_FACTORS - Standard 8 Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve RAW_FILE_NAME(File Name )=20240529_JV131_INVIVO_001_MalonicAcid_23 #COLLECTION CO:COLLECTION_SUMMARY Mice were sacrificed and liver tissue was collected. Tissues were flash frozen CO:COLLECTION_SUMMARY in liquid nitrogen and powderized using a mortar and pestle. Approximately 10 mg CO:COLLECTION_SUMMARY of tissue powder was transferred into an Eppendorf tube. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY 13C515N2-glutamine was resuspended to 50 mg/mL in 1X PBS. C57BL/6 mice between TR:TREATMENT_SUMMARY the ages of 10-16 weeks were weighed to allow injection of 0.75 g/kg. Mice that TR:TREATMENT_SUMMARY were injected with HKJS001 was resuspended in DMSO to have a final concentration TR:TREATMENT_SUMMARY of 3.33 mg/mL and 2uL of HKJS001 was injected per gram of mouse. Mice that were TR:TREATMENT_SUMMARY injected with malonic acid was resuspended in water to a molarity of 1M and TR:TREATMENT_SUMMARY 1.6uL of malonic acid was injected per gram of mouse. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY he tissue powder was re-suspended in 800 µL pre-cooled HPLC-grade 60:40 SP:SAMPLEPREP_SUMMARY Methanol:Water and then vortexed for 15 minutes at 4°C. 500 µL of pre-cooled SP:SAMPLEPREP_SUMMARY LC-MS grade chloroform was added to the lysate and again vortexed for 15 minutes SP:SAMPLEPREP_SUMMARY at 4°C. Samples were then centrifuged at 16,000 x g for 10 minutes at 4°C, SP:SAMPLEPREP_SUMMARY creating three layers: the top layer containing polar metabolites, the middle SP:SAMPLEPREP_SUMMARY layer containing protein, and the bottom layer containing non-polar metabolites. SP:SAMPLEPREP_SUMMARY The top layer was transferred into a new Eppendorf tube, dried down in a SP:SAMPLEPREP_SUMMARY Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 SP:SAMPLEPREP_SUMMARY Cold Trap, and stored at -80°C until they were re-suspended for LC-MS analysis. SP:SAMPLEPREP_SUMMARY The non-polar layer was discarded, and the remaining protein layer was saved for SP:SAMPLEPREP_SUMMARY protein quantification. #CHROMATOGRAPHY 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 20 min, 80% - 20% B; 0.5 min, 20% - 80% B; 7.5min, 80% B 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 POSITIVE MS:MS_COMMENTS The mass spectrometer was set to full scan (70-1000 m/z), with the spray voltage MS:MS_COMMENTS set to 4.0 kV, heated capillary to 350°C, and the HESI probe at 30 °C. The MS:MS_COMMENTS sheath gas flow was set at 10 units, auxiliary gas at 1 units, and sweep gas MS:MS_COMMENTS flow at 1 unit. The resolution of scan was set to 70,000, AGC target to 1x106, MS:MS_COMMENTS and maximum injection time at 20 msec. Data acquired by Thermo Fisher's Xcalibur MS:MS_COMMENTS software and analyzed by their Tracefinder software. The raw files provided MS:MS_COMMENTS contain data from both positive and negative ion mode. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples Liver 1 Liver 2 Liver 3 Liver 4 Liver 5 Liver 6 Liver 7 Liver 8 Liver 9 Liver 10 Liver 11 Liver 12 Liver 13 Liver 14 Liver 15 Liver 16 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Factors Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Vehicle Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Vehicle Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Vehicle Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Vehicle Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:HKJS001 Injected Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:HKJS001 Injected Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:HKJS001 Injected Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:HKJS001 Injected Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Malonic Acid Injected Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Malonic Acid Injected Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Malonic Acid Injected Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Malonic Acid Injected Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Combo HKJS001 and Malonic Acid Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Combo HKJS001 and Malonic Acid Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Combo HKJS001 and Malonic Acid Sample source:C57/BL6 mouse tissue | Standards for Standard Curve and Treatment:Combo HKJS001 and Malonic Acid Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve Sample source:Succinate and Fumarate Reagent Mixture | Standards for Standard Curve and Treatment:Measurable Standard Curve NAD+ 999456438 888514896 1122457931 1016907810 1545306100 1664363274 1072307587 1214364657 1225680567 1025128344 1154449106 964046535 1266669502 1139460035 1069741959 1336883309 980369.589 571061.483 418464.443 315094.82 234632.364 174684.447 136093.055 Glutathione 1.21E+10 1.19E+10 1.25E+10 1.55E+10 1.55E+10 1.32E+10 1.63E+10 1.50E+10 1.48E+10 1.16E+10 9199915008 1.43E+10 1.39E+10 1.38E+10 1.25E+10 1.55E+10 0 0 0 0 0 0 0 Oxidized glutathione 684318814 994113523 476466565 1051958878 1449501464 1439343729 1090664355 1270639936 1195938587 2726279487 1137008440 1037468672 718953986 940072131 992398793 1414610669 734155.554 192736.33 163259.794 148398.005 194045.43 141194.262 67322.5052 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention index Quantified m/z PubChem ID KEGG ID NAD+ 8.62 664.1164 5893 C00003 Glutathione 9.5 308.09108 124886 C00051 Oxidized glutathione 10.9 613.15924 65359 C00127 METABOLITES_END #END