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.
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.
Study ID | ST003192 |
Study Title | Aspartate tracing in Wildtype and UQCRC2 Knockout 143B Cells |
Study 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. 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 Name | Jerome |
First Name | Madison |
Address | 55 N Lake Ave, Worcester, MA 01655 |
madison.jerome@umassmed.edu | |
Phone | (508) 856-8989 ext. 68148 |
Submit Date | 2024-04-04 |
Num Groups | 3 |
Total Subjects | 2 |
Raw Data Available | Yes |
Raw Data File Type(s) | raw(Thermo) |
Analysis Type Detail | LC-MS |
Release Date | 2025-02-04 |
Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|
SA347900 | MJ01 | 143B osteosarcoma cells | DMSO |
SA347901 | MJ03 | 143B osteosarcoma cells | DMSO |
SA347902 | MJ02 | 143B osteosarcoma cells | DMSO |
SA347903 | MJ06 | 143B osteosarcoma cells | HKJS001 |
SA347904 | MJ05 | 143B osteosarcoma cells | HKJS001 |
SA347905 | MJ04 | 143B osteosarcoma cells | HKJS001 |
SA347906 | MJ09 | 143B osteosarcoma cells | HKJS003 |
SA347907 | MJ08 | 143B osteosarcoma cells | HKJS003 |
SA347908 | MJ07 | 143B 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 |