Summary of Study ST003404

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	ST003404
Study Title	Aspartate Tracing in SDHB KO RquA 143B Cells
Study Summary	143B cells that were cloned to have the SDHB(succinate dehydrogenase complex iron sulfur subunit B) KO and expression of the RquA enzyme were used to check for mitochondrial function of DHODH(dihydroorotate dehydrogenase). Cells were in culture for 4 days and then extracted for polar metabolites. Cells were treated with DMSO and brequinar. WT and RquA 143B cells were also used for comparison.
Institute	UMass Chan Medical School
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
Submit Date	2024-08-12
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-02-04
Release Version	1

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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:	SU003529
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	Cell Type	Treatment
SA371925	RquA Breq 2	143b Cells	RquA	Brequinar
SA371926	RquA Breq 3	143b Cells	RquA	Brequinar
SA371927	RquA Breq 1	143b Cells	RquA	Brequinar
SA371928	RquA DMSO 1	143b Cells	RquA	DMSO
SA371929	RquA DMSO 2	143b Cells	RquA	DMSO
SA371930	RquA DMSO 3	143b Cells	RquA	DMSO
SA371931	SDHB KO RquA Breq 1	143b Cells	SDHB KO RquA	Brequinar
SA371932	SDHB KO RquA Breq 2	143b Cells	SDHB KO RquA	Brequinar
SA371933	SDHB KO RquA Breq 3	143b Cells	SDHB KO RquA	Brequinar
SA371934	SDHB KO RquA DMSO 1	143b Cells	SDHB KO RquA	DMSO
SA371935	SDHB KO RquA DMSO 2	143b Cells	SDHB KO RquA	DMSO
SA371936	SDHB KO RquA DMSO 3	143b Cells	SDHB KO RquA	DMSO
SA371937	WT Breq 2	143b Cells	WT	Brequinar
SA371938	WT Breq 3	143b Cells	WT	Brequinar
SA371939	WT Breq 1	143b Cells	WT	Brequinar
SA371940	WT DMSO 2	143b Cells	WT	DMSO
SA371941	WT DMSO 3	143b Cells	WT	DMSO
SA371942	WT DMSO 1	143b Cells	WT	DMSO

Showing results 1 to 18 of 18

Showing results 1 to 18 of 18

Collection:

Collection ID:	CO003522
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. 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.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR003538
Treatment Summary:	Cells were seeded in complete DMEM 96 hours prior to tracing so that wells reached 75% confluence at time of experiment. DMEM containing 10% FBS, 1% penicillin and streptomycin, and 0.1 mg/mL uridine (Sigma) was used when seeding the experiment. 72 hours prior, the media was changed to DMEM containing 10% FBS, 1% penicillin and streptomycin, and 10 mM aspartate with the pH adjusted to 7.4 along with 1uL of either DMSO or 10mM of brequinar. 6 hours prior to metabolite isolation, the cells were treated with DMEM containing 10% FBS, 1% penicillin and streptomycin, and 10 mM 13C4-aspartate (Sigma) and the pH adjusted to 7.4 with the relevant treatments.

Treatment ID:

TR003538

Treatment Summary:

Cells were seeded in complete DMEM 96 hours prior to tracing so that wells reached 75% confluence at time of experiment. DMEM containing 10% FBS, 1% penicillin and streptomycin, and 0.1 mg/mL uridine (Sigma) was used when seeding the experiment. 72 hours prior, the media was changed to DMEM containing 10% FBS, 1% penicillin and streptomycin, and 10 mM aspartate with the pH adjusted to 7.4 along with 1uL of either DMSO or 10mM of brequinar. 6 hours prior to metabolite isolation, the cells were treated with DMEM containing 10% FBS, 1% penicillin and streptomycin, and 10 mM 13C4-aspartate (Sigma) and the pH adjusted to 7.4 with the relevant treatments.

Sample Preparation:

Sampleprep ID:	SP003536
Sampleprep Summary:	Once the samples were collected, they 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.

Sampleprep ID:

SP003536

Sampleprep Summary:

Once the samples were collected, they 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.

Combined analysis:

Analysis ID	AN005586	AN005587
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:	CH004246
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:	MS005311
Analysis ID:	AN005586
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:	MS005312
Analysis ID:	AN005587
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