#METABOLOMICS WORKBENCH spinelli_lab_20240419_071412 DATATRACK_ID:4785 STUDY_ID:ST003197 ANALYSIS_ID:AN005247 PROJECT_ID:PR001988 VERSION 1 CREATED_ON May 8, 2024, 10:09 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 wildtype mouse tissue ST:STUDY_SUMMARY The extraction of nonpolar metabolites from the tissues of wildtype mice for ST:STUDY_SUMMARY analysis by 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 Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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_KID01 Sample source:KIDNEY | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_rerun_01_20240408064230 SUBJECT_SAMPLE_FACTORS - MITO_KID03 Sample source:KIDNEY | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_01 SUBJECT_SAMPLE_FACTORS - MITO_KID04 Sample source:KIDNEY | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_02 SUBJECT_SAMPLE_FACTORS - MITO_KID05 Sample source:KIDNEY | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_03 SUBJECT_SAMPLE_FACTORS - MITO_KID06 Sample source:KIDNEY | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_04 SUBJECT_SAMPLE_FACTORS - MITO_KID07 Sample source:KIDNEY | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_05 SUBJECT_SAMPLE_FACTORS - MITO_B01 Sample source:BRAIN | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_06 SUBJECT_SAMPLE_FACTORS - MITO_B03 Sample source:BRAIN | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_07 SUBJECT_SAMPLE_FACTORS - MITO_B04 Sample source:BRAIN | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_08 SUBJECT_SAMPLE_FACTORS - MITO_B05 Sample source:BRAIN | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_09 SUBJECT_SAMPLE_FACTORS - MITO_B06 Sample source:BRAIN | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_10 SUBJECT_SAMPLE_FACTORS - MITO_B07 Sample source:BRAIN | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_11 SUBJECT_SAMPLE_FACTORS - MITO_P01 Sample source:PANCREAS | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_12 SUBJECT_SAMPLE_FACTORS - MITO_P03 Sample source:PANCREAS | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_13 SUBJECT_SAMPLE_FACTORS - MITO_P04 Sample source:PANCREAS | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_14 SUBJECT_SAMPLE_FACTORS - MITO_P05 Sample source:PANCREAS | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_15 SUBJECT_SAMPLE_FACTORS - MITO_P06 Sample source:PANCREAS | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_16 SUBJECT_SAMPLE_FACTORS - MITO_P07 Sample source:PANCREAS | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_17 SUBJECT_SAMPLE_FACTORS - MITO_H01 Sample source:HEART | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_18 SUBJECT_SAMPLE_FACTORS - MITO_H02 Sample source:HEART | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_19 SUBJECT_SAMPLE_FACTORS - MITO_H03 Sample source:HEART | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_20 SUBJECT_SAMPLE_FACTORS - MITO_H04 Sample source:HEART | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_21 SUBJECT_SAMPLE_FACTORS - MITO_H05 Sample source:HEART | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_22 SUBJECT_SAMPLE_FACTORS - MITO_H06 Sample source:HEART | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_23 SUBJECT_SAMPLE_FACTORS - MITO_LUNG01 Sample source:LUNG | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_24 SUBJECT_SAMPLE_FACTORS - MITO_LUNG03 Sample source:LUNG | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_25 SUBJECT_SAMPLE_FACTORS - MITO_LUNG04 Sample source:LUNG | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_26 SUBJECT_SAMPLE_FACTORS - MITO_LUNG05 Sample source:LUNG | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_27 SUBJECT_SAMPLE_FACTORS - MITO_LUNG06 Sample source:LUNG | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_28 SUBJECT_SAMPLE_FACTORS - MITO_LUNG07 Sample source:LUNG | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_29 SUBJECT_SAMPLE_FACTORS - MITO_M01 Sample source:MUSCLE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_30 SUBJECT_SAMPLE_FACTORS - MITO_M03 Sample source:MUSCLE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_31 SUBJECT_SAMPLE_FACTORS - MITO_M04 Sample source:MUSCLE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_32 SUBJECT_SAMPLE_FACTORS - MITO_M05 Sample source:MUSCLE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_33 SUBJECT_SAMPLE_FACTORS - MITO_M06 Sample source:MUSCLE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_34 SUBJECT_SAMPLE_FACTORS - MITO_M07 Sample source:MUSCLE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_35 SUBJECT_SAMPLE_FACTORS - MITO_IWAT01 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_36 SUBJECT_SAMPLE_FACTORS - MITO_IWAT03 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_37 SUBJECT_SAMPLE_FACTORS - MITO_IWAT04 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_38 SUBJECT_SAMPLE_FACTORS - MITO_IWAT05 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_39 SUBJECT_SAMPLE_FACTORS - MITO_IWAT06 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_40 SUBJECT_SAMPLE_FACTORS - MITO_IWAT07 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_41 SUBJECT_SAMPLE_FACTORS - MITO_LIV01 Sample source:LIVER | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_42 SUBJECT_SAMPLE_FACTORS - MITO_LIV03 Sample source:LIVER | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_43 SUBJECT_SAMPLE_FACTORS - MITO_LIV04 Sample source:LIVER | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_44 SUBJECT_SAMPLE_FACTORS - MITO_LIV05 Sample source:LIVER | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_45 SUBJECT_SAMPLE_FACTORS - MITO_LIV06 Sample source:LIVER | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_46 SUBJECT_SAMPLE_FACTORS - MITO_LIV07 Sample source:LIVER | Sample type:Mitochondria RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_47 SUBJECT_SAMPLE_FACTORS - WholeTissue_KID01 Sample source:KIDNEY | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_Mito_rerun_02_20240408065442 SUBJECT_SAMPLE_FACTORS - WholeTissue_KID03 Sample source:KIDNEY | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_01 SUBJECT_SAMPLE_FACTORS - WholeTissue_KID04 Sample source:KIDNEY | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_02 SUBJECT_SAMPLE_FACTORS - WholeTissue_KID05 Sample source:KIDNEY | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_03 SUBJECT_SAMPLE_FACTORS - WholeTissue_KID06 Sample source:KIDNEY | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_04 SUBJECT_SAMPLE_FACTORS - WholeTissue_KID07 Sample source:KIDNEY | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_05 SUBJECT_SAMPLE_FACTORS - WholeTissue_B01 Sample source:BRAIN | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_06 SUBJECT_SAMPLE_FACTORS - WholeTissue_B03 Sample source:BRAIN | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_07 SUBJECT_SAMPLE_FACTORS - WholeTissue_B04 Sample source:BRAIN | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_08 SUBJECT_SAMPLE_FACTORS - WholeTissue_B05 Sample source:BRAIN | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_09 SUBJECT_SAMPLE_FACTORS - WholeTissue_B06 Sample source:BRAIN | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_10 SUBJECT_SAMPLE_FACTORS - WholeTissue_B07 Sample source:BRAIN | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_11 SUBJECT_SAMPLE_FACTORS - WholeTissue_P01 Sample source:PANCREAS | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_12 SUBJECT_SAMPLE_FACTORS - WholeTissue_P03 Sample source:PANCREAS | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_13 SUBJECT_SAMPLE_FACTORS - WholeTissue_P04 Sample source:PANCREAS | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_14 SUBJECT_SAMPLE_FACTORS - WholeTissue_P05 Sample source:PANCREAS | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_15 SUBJECT_SAMPLE_FACTORS - WholeTissue_P06 Sample source:PANCREAS | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_16 SUBJECT_SAMPLE_FACTORS - WholeTissue_P07 Sample source:PANCREAS | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_17 SUBJECT_SAMPLE_FACTORS - WholeTissue_H01 Sample source:HEART | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_18 SUBJECT_SAMPLE_FACTORS - WholeTissue_H03 Sample source:HEART | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_19 SUBJECT_SAMPLE_FACTORS - WholeTissue_H04 Sample source:HEART | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_20 SUBJECT_SAMPLE_FACTORS - WholeTissue_H05 Sample source:HEART | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_21 SUBJECT_SAMPLE_FACTORS - WholeTissue_H06 Sample source:HEART | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_22 SUBJECT_SAMPLE_FACTORS - WholeTissue_H07 Sample source:HEART | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_23 SUBJECT_SAMPLE_FACTORS - WholeTissue_LUNG01 Sample source:LUNG | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_24 SUBJECT_SAMPLE_FACTORS - WholeTissue_LUNG03 Sample source:LUNG | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_25 SUBJECT_SAMPLE_FACTORS - WholeTissue_LUNG04 Sample source:LUNG | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_26 SUBJECT_SAMPLE_FACTORS - WholeTissue_LUNG05 Sample source:LUNG | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_27 SUBJECT_SAMPLE_FACTORS - WholeTissue_LUNG06 Sample source:LUNG | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_28 SUBJECT_SAMPLE_FACTORS - WholeTissue_LUNG07 Sample source:LUNG | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_29 SUBJECT_SAMPLE_FACTORS - WholeTissue_M01 Sample source:MUSCLE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_30 SUBJECT_SAMPLE_FACTORS - WholeTissue_M03 Sample source:MUSCLE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_31 SUBJECT_SAMPLE_FACTORS - WholeTissue_M04 Sample source:MUSCLE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_32 SUBJECT_SAMPLE_FACTORS - WholeTissue_M05 Sample source:MUSCLE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_33 SUBJECT_SAMPLE_FACTORS - WholeTissue_M06 Sample source:MUSCLE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_34 SUBJECT_SAMPLE_FACTORS - WholeTissue_M07 Sample source:MUSCLE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_35 SUBJECT_SAMPLE_FACTORS - WholeTissue_IWAT01 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_36 SUBJECT_SAMPLE_FACTORS - WholeTissue_IWAT03 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_37 SUBJECT_SAMPLE_FACTORS - WholeTissue_IWAT04 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_38 SUBJECT_SAMPLE_FACTORS - WholeTissue_IWAT05 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_39 SUBJECT_SAMPLE_FACTORS - WholeTissue_IWAT06 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_40 SUBJECT_SAMPLE_FACTORS - WholeTissue_IWAT07 Sample source:INGUINAL WHITE ADIPOSE TISSUE | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_41 SUBJECT_SAMPLE_FACTORS - WholeTissue_LIV01 Sample source:LIVER | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_42 SUBJECT_SAMPLE_FACTORS - WholeTissue_LIV03 Sample source:LIVER | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_43 SUBJECT_SAMPLE_FACTORS - WholeTissue_LIV04 Sample source:LIVER | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_44 SUBJECT_SAMPLE_FACTORS - WholeTissue_LIV05 Sample source:LIVER | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_45 SUBJECT_SAMPLE_FACTORS - WholeTissue_LIV06 Sample source:LIVER | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_46 SUBJECT_SAMPLE_FACTORS - WholeTissue_LIV07 Sample source:LIVER | Sample type:Whole Tissue RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_47 SUBJECT_SAMPLE_FACTORS - CoQ9_01 Sample source:CoQ9 0.001µM Standard | Sample type:CoQ9 Standard RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_48 SUBJECT_SAMPLE_FACTORS - CoQ9_02 Sample source:CoQ9 0.01µM Standard | Sample type:CoQ9 Standard RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_49 SUBJECT_SAMPLE_FACTORS - CoQ9_03 Sample source:CoQ9 0.1µM Standard | Sample type:CoQ9 Standard RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_50 SUBJECT_SAMPLE_FACTORS - CoQ9_04 Sample source:CoQ9 0.25µM Standard | Sample type:CoQ9 Standard RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_51 SUBJECT_SAMPLE_FACTORS - CoQ9_05 Sample source:CoQ9 0.5µM Standard | Sample type:CoQ9 Standard RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_52 SUBJECT_SAMPLE_FACTORS - CoQ9_06 Sample source:CoQ9 1µM Standard | Sample type:CoQ9 Standard RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_53 SUBJECT_SAMPLE_FACTORS - CoQ9_07 Sample source:CoQ9 5µM Standard | Sample type:CoQ9 Standard RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_54 SUBJECT_SAMPLE_FACTORS - CoQ9_08 Sample source:CoQ9 10µM Standard | Sample type:CoQ9 Standard RAW_FILE_NAME(Raw file name)=20240405_MJ45_WholeTissue_55 #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 Kidney, Brain, Pancreas, Heart, Lung, Muscle, inguinal white adipose tissue & CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Wildtype 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. The raw files MS:MS_COMMENTS provided contain data from both positive and negative ion mode. The data MS:MS_COMMENTS reported includes the different adducts and reduced forms of the metabolites to MS:MS_COMMENTS show presence or lack thereof. MS:MS_RESULTS_FILE ST003197_AN005247_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END