Summary of Study ST003218
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 | ST003218 |
| Study Title | Glutamine Tracing Assay in WT and SDHB KO 143B cells Treated with HKJS003 |
| Study Summary | WT and succinate dehydrogenase iron sulfur subunit B(SDHB) KO 143B cells were treated with a small molecule analog of Rhodoquinone, HKJS003. Glutamine tracing was used to determine the fumarate reduction and succinate oxidation of the treated cells. Other metabolites such as ATP,ADP,AMP,Glutathione, Oxidized glutathione, UTP, lactic acid, and pyruvic acid were also measured. |
| 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: | SU003337 |
| 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 |
|---|---|---|---|
| SA351818 | SDHB KO 100nM 003 2 | SDHB KO | 100nM HKJS003 |
| SA351819 | SDHB KO 100nM 003 1 | SDHB KO | 100nM HKJS003 |
| SA351820 | SDHB KO 100nM 003 3 | SDHB KO | 100nM HKJS003 |
| SA351821 | SDHB KO 25nM 003 1 | SDHB KO | 25nM HKJS003 |
| SA351822 | SDHB KO 25nM 003 2 | SDHB KO | 25nM HKJS003 |
| SA351823 | SDHB KO 25nM 003 3 | SDHB KO | 25nM HKJS003 |
| SA351824 | SDHB KO 3 | SDHB KO | DMSO |
| SA351825 | SDHB KO 2 | SDHB KO | DMSO |
| SA351826 | SDHB KO 1 | SDHB KO | DMSO |
| SA351827 | WT 100nM 003 1 | WT | 100nM HKJS003 |
| SA351828 | WT 100nM 003 2 | WT | 100nM HKJS003 |
| SA351829 | WT 100nM 003 3 | WT | 100nM HKJS003 |
| SA351830 | WT 25nM 003 1 | WT | 25nM HKJS003 |
| SA351831 | WT 25nM 003 2 | WT | 25nM HKJS003 |
| SA351832 | WT 25nM 003 3 | WT | 25nM HKJS003 |
| SA351833 | WT 2 | WT | DMSO |
| SA351834 | WT 3 | WT | DMSO |
| SA351835 | WT 1 | WT | DMSO |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO003330 |
| Collection Summary: | Media was aspirated from the plates and then the cells were washed with 1x PBS twice. The plate was then transferred to dry ice and 500 µL of 80% HPLC-grade methanol (Sigma) 20% HPLC-grade water (Sigma) was added to each well. The wells were placed in a -80 freezer to incubate for 15 minutes. The plates are taken out of the freezer one at a time and placed back on dry ice. The cells were then scraped and transferred to a new tube. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003346 |
| 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.Cells were then supplemented with either DMSO or HKJS003. For HKJS003, 20µL or 5µL of 100µM stock was added to 2mL of media for a final concentration of 100nM or 25nM. For DMSO, 20µL of a diluted 1:1000 DMSO was added to 2mL of media. The cells were incubated for 5-7 of days to proliferate and every 2 days the media was refreshed with the relevant treatments. |
Sample Preparation:
| Sampleprep ID: | SP003344 |
| Sampleprep Summary: | Once cells were collected into Eppendorf tubes, each well was washed with an additional 300 µL of 80% HPLC-grade methanol (Sigma) 20% HPLC-grade water (Sigma) and collected into the same tube as the initial lysis. The samples were then vortexed at 4° C for 10 minutes and centrifuged at 21,300 x g for 10 minutes at 4° C. Supernatants were transferred to a new tube and dried down in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). After being dried down, pellets were stored in a -20° C freezer until ready to run on the polar LC-MS method. |
Chromatography:
| Chromatography ID: | CH003992 |
| Chromatography Summary: | 20 min, 80% to 20% B; 0.5 min, 20% to 80% B; 7.5min, 80% B |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| Column Temperature: | 25 |
| Flow Gradient: | Gradient starts at 80% B. Subsequently, 80% to 20% B linearly in 20min; 20% to 80% B in 0.5min; 80% B for 7.5min |
| Flow Rate: | 0.15ml/min |
| Solvent A: | 100% water; 0.1% ammonium hydroxide; 20mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
Analysis:
| Analysis ID: | AN005276 |
| Analysis Type: | MS |
| Chromatography ID: | CH003992 |
| Num Factors: | 6 |
| Num Metabolites: | 2 |
| Rt Units: | Minutes |
| Units: | peak area |
| Analysis ID: | AN005277 |
| Analysis Type: | MS |
| Chromatography ID: | CH003992 |
| Num Factors: | 6 |
| Num Metabolites: | 16 |
| Rt Units: | Minutes |
| Units: | peak area |
