Summary of Study ST003217
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 | ST003217 |
| Study Title | Measuring UQ/UQH2 in WT and RquA 143B cell in Hypoxia |
| Study Summary | Cultured cells were used to measure the ratio of UQ to UQH2 in WT and RquA 143B cells. Cells were exposed to hypoxia to see if there was a difference in UQ:UQH2 ratio. |
| Institute | UMass Chan Medical School |
| Last Name | UMass Chan |
| First Name | Spinelli Lab |
| Address | 55 Lake Avenue North, Worcester, Massachusetts, 01605, USA |
| spinellilab@gmail.com | |
| Phone | (508) 856-8989 ext. 68148 |
| Submit Date | 2024-05-20 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, 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: | SU003336 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Treatment |
|---|---|---|---|
| SA351806 | RquA Hypoxia 1 | RquA expressing | hypoxia exposed |
| SA351807 | RquA Hypoxia 2 | RquA expressing | hypoxia exposed |
| SA351808 | RquA Hypoxia 3 | RquA expressing | hypoxia exposed |
| SA351809 | RquA 3 | RquA expressing | normoxia |
| SA351810 | RquA 2 | RquA expressing | normoxia |
| SA351811 | RquA 1 | RquA expressing | normoxia |
| SA351812 | WT Hypoxia 1 | WT | hypoxia exposed |
| SA351813 | WT Hypoxia 2 | WT | hypoxia exposed |
| SA351814 | WT Hypoxia 3 | WT | hypoxia exposed |
| SA351815 | WT 2 | WT | normoxia |
| SA351816 | WT 3 | WT | normoxia |
| SA351817 | WT 1 | WT | normoxia |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO003329 |
| Collection Summary: | Plates were removed from incubator, media was aspirated, wells were washed with 1X PBS, and the wash was aspirated. Plates were moved to dry ice and 500 µL of 100% LCMS-grade ethanol (Sigma) was added to each well. Cells were then scraped with cell scraper and lysate was moved to a labeled and pre-cooled 1.5 mL Eppendorf tube. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003345 |
| Treatment Summary: | The 143B cell line were cultured in Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher) supplemented with 10% Heat Inactivated Fetal Bovine Serum (ThermoFisher) and 1% penicillin and streptomycin (ThermoFisher), and 100 µg/mL uridine (Sigma). Cells were kept in a 37°C incubator (Baker Ruskinn) held at 5% CO2 and 90% relative humidity. 25,000 – 50,000 cells were seeded in complete DMEM media. 24 hours later the media was changed with 1.25µL of 10mg/mL of doxycycline in 50mL of DMEM to induce RquA expression. The cells were incubated for 5-7 of days to proliferate and every 2 days the media was refreshed. For conditions done in hypoxia, cells were initially seeded in normoxia. 24 hours after seeding, the cells are transferred into the Invivo2 1000 workstation (Baker Ruskinn) with the oxygen tension set to 0.5% O2, the temperature to 37°C, the CO2 to 5%, and the relative humidity to 80%. Media would be changed in the Invivo2 1000 workstation the same as normoxia. |
Sample Preparation:
| Sampleprep ID: | SP003343 |
| Sampleprep Summary: | Once cells were collected into Eppendorf tubes,samples were then vortexed for 10 minutes in a cold room. Then, 1 mL of 100% LCMS-grade hexane (sigma) added to it and samples were vortexed for another 10 minutes in the cold room. Samples were 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. |
Chromatography:
| Chromatography ID: | CH003991 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Luna PFP(2) (100 x 2mm, 3um) |
| 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 |
Analysis:
| Analysis ID: | AN005275 |
| Analysis Type: | MS |
| Chromatography ID: | CH003991 |
| Has Mz: | 1 |
| Has Rt: | 1 |
| Rt Units: | Minutes |
| Results File: | ST003217_AN005275_Results.txt |
| Units: | peak area |
