Summary of Study ST003204
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 | ST003204 |
| Study Title | Glutamine Tracing Assay on Caki1 WT and 769P WT versus Caki1 RquA and 769P RquA |
| Study Summary | The extraction of Caki1 and 769P cell types in the condition WT versus RquA and in 21% O2 versus 0.5% O2 for 13C5 Glutamine Tracing Assay. |
| 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 | 2024-05-08 |
| 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: | SU003323 |
| 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 | Sample source | Treatment |
|---|---|---|---|
| SA348784 | RQUA 769P 0.5% 1 | Human Renal 769P Cells | RquA 0.5% O2 |
| SA348785 | RQUA 769P 0.5% 2 | Human Renal 769P Cells | RquA 0.5% O2 |
| SA348786 | RQUA 769P 0.5% 3 | Human Renal 769P Cells | RquA 0.5% O2 |
| SA348787 | RQUA 769P 21% 2 | Human Renal 769P Cells | RquA 21% O2 |
| SA348788 | RQUA 769P 21% 1 | Human Renal 769P Cells | RquA 21% O2 |
| SA348789 | RQUA 769P 21% 3 | Human Renal 769P Cells | RquA 21% O2 |
| SA348790 | WT 769P 0.5% 3 | Human Renal 769P Cells | WT 0.5% O2 |
| SA348791 | WT 769P 0.5% 1 | Human Renal 769P Cells | WT 0.5% O2 |
| SA348792 | WT 769P 0.5% 2 | Human Renal 769P Cells | WT 0.5% O2 |
| SA348793 | WT 769P 21% 3 | Human Renal 769P Cells | WT 21% O2 |
| SA348794 | WT 769P 21% 2 | Human Renal 769P Cells | WT 21% O2 |
| SA348795 | WT 769P 21% 1 | Human Renal 769P Cells | WT 21% O2 |
| SA348796 | RQUA CAKI 1 0.5% 1 | Human Renal Caki1 Cells | RquA 0.5% O2 |
| SA348797 | RQUA CAKI 1 0.5% 2 | Human Renal Caki1 Cells | RquA 0.5% O2 |
| SA348798 | RQUA CAKI 1 0.5% 3 | Human Renal Caki1 Cells | RquA 0.5% O2 |
| SA348799 | RQUA CAKI 1 21% 2 | Human Renal Caki1 Cells | RquA 21% O2 |
| SA348800 | RQUA CAKI 1 21% 1 | Human Renal Caki1 Cells | RquA 21% O2 |
| SA348801 | RQUA CAKI 1 21% 3 | Human Renal Caki1 Cells | RquA 21% O2 |
| SA348802 | WT CAKI 1 0.5% 2 | Human Renal Caki1 Cells | WT 0.5% O2 |
| SA348803 | WT CAKI 1 0.5% 1 | Human Renal Caki1 Cells | WT 0.5% O2 |
| SA348804 | WT CAKI 1 0.5% 3 | Human Renal Caki1 Cells | WT 0.5% O2 |
| SA348805 | WT CAKI 1 21% 2 | Human Renal Caki1 Cells | WT 21% O2 |
| SA348806 | WT CAKI 1 21% 3 | Human Renal Caki1 Cells | WT 21% O2 |
| SA348807 | WT CAKI 1 21% 1 | Human Renal Caki1 Cells | WT 21% O2 |
| Showing results 1 to 24 of 24 |
Collection:
| Collection ID: | CO003316 |
| Collection Summary: | Human Renal Caki-1 Cells and Human Renal 769P Cells were purchased from ATCC and cultured in complete DMEM media. When the cells were collected the media was aspirated from the plates and then the cells were washed with 1x PBS (Phosphate Buffered Saline) twice to wash the cells of old media. 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. 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. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003332 |
| Treatment Summary: | Cells were seeded in complete DMEM 5 days prior to tracing so that wells reached 75% confluence at the time of the experiment. After seeding, cells were treated with their respective conditions (WT versus RquA) and incubated in 21% O2 or 0.5% O2 for seven days. For experiments involving the hypoxia chamber (0.5% O2), media were preconditioned at 0.5% O2 for 72 hours prior to use for experiments. Tracing media was then prepared, with glutamine-free DMEM (Gibco) containing 10% Fetal Bovine Serum (FBS), 1% PenStrep, and 0.1 mg/mL uridine supplemented with 2 mM 13C515N2-glutamine (Cambridge Isotope Labs). Media was refreshed in each well with stable isotope tracing media and incubated for 8 hours. |
Sample Preparation:
| Sampleprep ID: | SP003330 |
| Sampleprep Summary: | The pellet was resuspended in 200uL of LCMS H2O (Sigma), vortexed for 10 minutes in 4°C, centrifuged for 10 minutes at 21,300 x g at 4°C, and then moved 20uL into LCMS vials to run on the LCMS. |
Combined analysis:
| Analysis ID | AN005255 | AN005256 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) | Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH003978 |
| 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: | MS004987 |
| Analysis ID: | AN005255 |
| 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. 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: | POSITIVE |
| MS ID: | MS004988 |
| Analysis ID: | AN005256 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The mass spectrometer had 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. 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 |
