Summary of Study ST003198

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001988. The data can be accessed directly via it's Project DOI: 10.21228/M8RJ07 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003198
Study TitleUQ/RQ panel on human tissue
Study SummaryThe 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
DepartmentProgram in Molecular Medicine
LaboratorySpinelli Lab
Last NameJerome
First NameMadison
Address55 N Lake Ave, Worcester, MA 01655
Emailmadison.jerome@umassmed.edu
Phone(508) 856-8989 ext. 68148
Submit Date2024-05-01
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2025-02-04
Release Version1
Madison Jerome Madison Jerome
https://dx.doi.org/10.21228/M8RJ07
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001988
Project DOI:doi: 10.21228/M8RJ07
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:UMass Chan
First Name:Spinelli Lab
Address:55 Lake Avenue North, Worcester, Massachusetts, 01605, USA
Email:spinellilab@gmail.com
Phone:(508) 856-8989 ext. 68148

Subject:

Subject ID:SU003317
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Sample type
SA348075Mito AP01Adipose Pannus Mitochondria
SA348076Mito AP02Adipose Pannus Mitochondria
SA348077Mito AP03Adipose Pannus Mitochondria
SA348078Mito AP05Adipose Pannus Mitochondria
SA348079Mito AP04Adipose Pannus Mitochondria
SA348080WholeTissue AP01Adipose Pannus Whole Tissue
SA348081WholeTissue AP03Adipose Pannus Whole Tissue
SA348082WholeTissue AP02Adipose Pannus Whole Tissue
SA348083WholeTissue AP05Adipose Pannus Whole Tissue
SA348084WholeTissue AP04Adipose Pannus Whole Tissue
SA348085Mito B04Brain Mitochondria
SA348086Mito B03Brain Mitochondria
SA348087Mito B02Brain Mitochondria
SA348088Mito B01Brain Mitochondria
SA348089Mito B05Brain Mitochondria
SA348090WholeTissue B02Brain Whole Tissue
SA348091WholeTissue B04Brain Whole Tissue
SA348092WholeTissue B03Brain Whole Tissue
SA348093WholeTissue B01Brain Whole Tissue
SA348094WholeTissue B05Brain Whole Tissue
SA348095Mito K01Kidney Mitochondria
SA348096Mito K02Kidney Mitochondria
SA348097Mito K03Kidney Mitochondria
SA348098Mito K05Kidney Mitochondria
SA348099Mito K04Kidney Mitochondria
SA348100WholeTissue K05Kidney Whole Tissue
SA348101WholeTissue K04Kidney Whole Tissue
SA348102WholeTissue K03Kidney Whole Tissue
SA348103WholeTissue K01Kidney Whole Tissue
SA348104WholeTissue K02Kidney Whole Tissue
SA348105Mito M01Muscle Mitochondria
SA348106Mito M05Muscle Mitochondria
SA348107Mito M02Muscle Mitochondria
SA348108Mito M03Muscle Mitochondria
SA348109Mito M04Muscle Mitochondria
SA348110WholeTissue M05Muscle Whole Tissue
SA348111WholeTissue M04Muscle Whole Tissue
SA348112WholeTissue M02Muscle Whole Tissue
SA348113WholeTissue M01Muscle Whole Tissue
SA348114WholeTissue M03Muscle Whole Tissue
SA348115CoQ10 5µMStandard CoQ10 Standard
SA348116CoQ10 10µMStandard CoQ10 Standard
SA348117CoQ10 1µMStandard CoQ10 Standard
SA348118CoQ10 0.1µMStandard CoQ10 Standard
SA348119CoQ10 0.001µMStandard CoQ10 Standard
SA348120CoQ10 0.01µMStandard CoQ10 Standard
SA348121CoQ10 0.25µMStandard CoQ10 Standard
SA348122CoQ10 0.5µMStandard CoQ10 Standard
Showing results 1 to 48 of 48

Collection:

Collection ID:CO003310
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 ID:TR003326
Treatment Summary:Untreated

Sample Preparation:

Sampleprep ID:SP003324
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.

Combined analysis:

Analysis ID AN005248
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Vanquish
Column Phenomenex Luna PFP(2) (100 x 2mm,3µm)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode POSITIVE
Units peak area

Chromatography:

Chromatography ID:CH003973
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Luna PFP(2) (100 x 2mm,3µm)
Column Temperature:25
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
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS004981
Analysis ID:AN005248
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
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.
Ion Mode:POSITIVE
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