Summary of Study ST003192

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.

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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 IDST003192
Study TitleAspartate tracing in Wildtype and UQCRC2 Knockout 143B Cells
Study SummaryUbiquinone (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. Wildtype 143B and UQCRC2 knockout cells were seeded and treated with media containing 13C4-Aspartate. The cells were extracted for metabolomics and ran on the LC/MS on the pHILIC method.
Institute
UMass Chan Medical School
Last NameJerome
First NameMadison
Address55 N Lake Ave, Worcester, MA 01655
Emailmadison.jerome@umassmed.edu
Phone(508) 856-8989 ext. 68148
Submit Date2024-04-04
Num Groups3
Total Subjects2
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:SU003311
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Cell Strain Details:143B
Species Group:Mammals

Factors:

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

mb_sample_id local_sample_id Sample source Treatment
SA347900MJ01143B osteosarcoma cells DMSO
SA347901MJ03143B osteosarcoma cells DMSO
SA347902MJ02143B osteosarcoma cells DMSO
SA347903MJ06143B osteosarcoma cells HKJS001
SA347904MJ05143B osteosarcoma cells HKJS001
SA347905MJ04143B osteosarcoma cells HKJS001
SA347906MJ09143B osteosarcoma cells HKJS003
SA347907MJ08143B osteosarcoma cells HKJS003
SA347908MJ07143B osteosarcoma cells HKJS003
Showing results 1 to 9 of 9

Collection:

Collection ID:CO003304
Collection Summary:Cells were cultured in DMEM with 10% FBS and 1% Pen Strep and treated with either DMSO, 10uM HKJS001, or 25nM HKJS003. The media was changed after two days to media containing 13C4-aspartate and the treatments were added again. The cells were incubated with the 13C4-aspartate media at 37 degrees Celsius for 6 hours before being isolated. The cells were taken out of the incubator and placed on water ice where the media was aspirated off, washed twice with 1X PBS, and treated with 500uL of 80% LCMS grade MeOH per well on dry ice. The cells were incubated in the -80 degrees Celsius freezer for at least 15 minutes. The cells were lifted using cell scrapers on dry ice and the well was washed with an additional 300uL of 80% LCMS grade MeOH. The cell solution was transferred to a 1.5mL Eppendorf tube vortexed for 10 minutes at 4 degrees Celsius in a cold room. The tubes were then centrifuged at 21300rcf at 4 degrees Celsius for 10 minutes. The supernatant was transferred to a new tube, and the sample was dried down using a speed vac to dry the pellet.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR003320
Treatment Summary:143B cells were treated with each media change. When new media was added the cells either received DMSO as a control, 10uM of small molecule HKJS001, or 25nM of small molecule HKJS003 with three replicates for each treatment. The plates were swirled gently to mix the treatment into the media.

Sample Preparation:

Sampleprep ID:SP003318
Sampleprep Summary:The samples (dried down pellets) were resuspended in 100uL of LCMS grade water and vortexed for 10 minutes at 4 degrees Celsius in a cold room. They were then centrifuged for 10 minutes at 4 degrees Celsius at 21300rcf. 20uL of each sample was transferred to a LCMS vial.
Extract Storage:-80℃
Sample Resuspension:100uL

Combined analysis:

Analysis ID AN005240
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode NEGATIVE
Units Peak area

Chromatography:

Chromatography ID:CH003967
Instrument Name:Thermo Vanquish
Column Name:Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
Column Temperature:25
Flow Gradient:20 min, 80% - 20% B; 0.5 min, 20% - 80% B; 7.5min, 80% B
Flow Rate:0.15ml/min
Solvent A:100% water; 0.1% ammonium hydroxide; 20mM ammonium carbonate
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS004973
Analysis ID:AN005240
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The mass spectrometer was set to full scan (70-1000 m/z), with the spray voltage set to 4.0 kV, heated capillary to 350°C, and the HESI probe at 30 °C. The sheath gas flow was set at 10 units, auxiliary gas at 1 units, and sweep gas flow at 1 unit. The resolution of scan was set to 70,000, AGC target to 1x106, and maximum injection time at 20 msec. An additional scan between 220-700 m/z was used to enhance nucleotide detection in the negative mode as well with the maximum injection time set to 80 msec. Data acquired by Thermo Fisher's Xcalibur software and analyzed by their Tracefinder software. The raw files provided contain data from both positive and negative ion mode.
Ion Mode:NEGATIVE
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