Summary of Study ST003214

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	ST003214
Study Title	Glutamine Tracing Assay in RquA AAV Injected Mice with a Standard Curve
Study Summary	The extraction of RquA AAV injected livers from C57BL/6 mice. Metabolites from the liver were used to measure fumarate reduction and succinate oxidation. Glutamine tracing was used to determine forward and reverse direction of the TCA cycle. Standards were used to create a standard curve by diluting down a stock of fumarate and succinate standard mixture. The standard curve was used to determine picomoles of metabolites in the TCA cycle.
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	2024-05-14
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:	SU003333
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammals

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Standards for Standard Curve and Treatment
SA351717	Liver 2	C57/BL6 mouse liver tissue	RquA AAV injected
SA351718	Liver 3	C57/BL6 mouse liver tissue	RquA AAV injected
SA351719	Liver 6	C57/BL6 mouse liver tissue	RquA AAV injected
SA351720	Liver 1	C57/BL6 mouse liver tissue	RquA AAV injected
SA351721	Liver 5	C57/BL6 mouse liver tissue	RquA AAV injected
SA351722	Liver 4	C57/BL6 mouse liver tissue	RquA AAV injected
SA351723	Liver 11	C57/BL6 mouse liver tissue	Vehicle
SA351724	Liver 10	C57/BL6 mouse liver tissue	Vehicle
SA351725	Liver 12	C57/BL6 mouse liver tissue	Vehicle
SA351726	Liver 13	C57/BL6 mouse liver tissue	Vehicle
SA351727	Liver 15	C57/BL6 mouse liver tissue	Vehicle
SA351728	Liver 9	C57/BL6 mouse liver tissue	Vehicle
SA351729	Liver 8	C57/BL6 mouse liver tissue	Vehicle
SA351730	Liver 7	C57/BL6 mouse liver tissue	Vehicle
SA351731	Standard 7	Succinate and Fumarate Reagent Mixture	Measurable Standard Curve
SA351732	Standard 3	Succinate and Fumarate Reagent Mixture	Measurable Standard Curve
SA351733	Standard 6	Succinate and Fumarate Reagent Mixture	Measurable Standard Curve
SA351734	Standard 5	Succinate and Fumarate Reagent Mixture	Measurable Standard Curve
SA351735	Standard 4	Succinate and Fumarate Reagent Mixture	Measurable Standard Curve
SA351736	Standard 8	Succinate and Fumarate Reagent Mixture	Measurable Standard Curve
SA351737	Standard 2	Succinate and Fumarate Reagent Mixture	Measurable Standard Curve

Showing results 1 to 21 of 21

Showing results 1 to 21 of 21

Collection:

Collection ID:	CO003326
Collection Summary:	13C515N2-glutamine (Cambridge Isotope labs) was resuspended to 50 mg/mL in 1X PBS. C57BL/6 mice between the ages of 10-16 weeks were weighed to allow injection of 0.75 g/kg intraperitoneally and 0.2 g/kg intravenously via the tail vein. Mice were injected with the respective amounts of 13C515N2-glutamine, were sacrificed after 45 minutes, and tissues were harvested and flash frozen in Liquid Nitrogen. Samples were stored at -80°C until their extraction and run on the polar LC-MS method.
Sample Type:	Mouse Liver

Treatment:

Treatment ID:	TR003342
Treatment Summary:	Adeno-associated viruses (AAVs) were used for expression of RquA in mice. Briefly, rAAV was produced by transient HEK 293 cell transfection and CsCl sedimentation by the University of Massachusetts Medical School Viral Vector Core. Vector genome titers of the rAAV preparations for control virus (AAV9.Tre3G-mCherry SV40 CB6 Tet-on 3G RBG) and RquA virus (AAV9.Tre3G-RquA SV40 CB6 Tet-on 3G RBG) were determined by ddPCR, and purity was assessed by 4%–12% SDS-acrylamide gel electrophoresis and silver staining (Invitrogen). Mice were intravenously injected with 6 X 10^11 genome copies of either virus and given 1 mg/mL doxycycline in the drinking water for 7 days to induce either mCherry or RquA expression.

Treatment ID:

TR003342

Treatment Summary:

Adeno-associated viruses (AAVs) were used for expression of RquA in mice. Briefly, rAAV was produced by transient HEK 293 cell transfection and CsCl sedimentation by the University of Massachusetts Medical School Viral Vector Core. Vector genome titers of the rAAV preparations for control virus (AAV9.Tre3G-mCherry SV40 CB6 Tet-on 3G RBG) and RquA virus (AAV9.Tre3G-RquA SV40 CB6 Tet-on 3G RBG) were determined by ddPCR, and purity was assessed by 4%–12% SDS-acrylamide gel electrophoresis and silver staining (Invitrogen). Mice were intravenously injected with 6 X 10^11 genome copies of either virus and given 1 mg/mL doxycycline in the drinking water for 7 days to induce either mCherry or RquA expression.

Sample Preparation:

Sampleprep ID:	SP003340
Sampleprep Summary:	Tissues were flash frozen in liquid nitrogen and powderized using a mortar and pestle. Approximately 10 mg of tissue powder was transferred into an Eppendorf tube. The tissue powder was re-suspended in 800 µL pre-cooled HPLC-grade 60:40 Methanol:Water (Sigma) and then vortexed for 15 minutes at 4°C. 500 µL of pre-cooled LC-MS grade chloroform (Sigma) was added to the lysate and again vortexed for 15 minutes at 4°C. Samples were then centrifuged at 16,000 x g for 10 minutes at 4°C, creating three layers: the top layer containing polar metabolites, the middle layer containing protein, and the bottom layer containing non-polar metabolites. The top layer was transferred into a new Eppendorf tube, dried down in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco), and stored at -80°C until they were re-suspended for LC-MS analysis. The non-polar layer was discarded, and the remaining protein layer was saved for protein quantification. The protein layer from the metabolite isolation was re-suspended in 1 mL of RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 0.1% SDS, 1% Triton-X 100 (Sigma), 0.5% deoxycholate (Sigma), cOmplete EDTA-free protease inhibitor (Sigma)). The samples were vortexed for 10 minutes at 4°C and then centrifuged at 16,000 x g for 10 minutes at 4°C. Protein concentrations for each sample were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (1 µg/µL ) concentration.

Sampleprep ID:

SP003340

Sampleprep Summary:

Tissues were flash frozen in liquid nitrogen and powderized using a mortar and pestle. Approximately 10 mg of tissue powder was transferred into an Eppendorf tube. The tissue powder was re-suspended in 800 µL pre-cooled HPLC-grade 60:40 Methanol:Water (Sigma) and then vortexed for 15 minutes at 4°C. 500 µL of pre-cooled LC-MS grade chloroform (Sigma) was added to the lysate and again vortexed for 15 minutes at 4°C. Samples were then centrifuged at 16,000 x g for 10 minutes at 4°C, creating three layers: the top layer containing polar metabolites, the middle layer containing protein, and the bottom layer containing non-polar metabolites. The top layer was transferred into a new Eppendorf tube, dried down in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco), and stored at -80°C until they were re-suspended for LC-MS analysis. The non-polar layer was discarded, and the remaining protein layer was saved for protein quantification. The protein layer from the metabolite isolation was re-suspended in 1 mL of RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 0.1% SDS, 1% Triton-X 100 (Sigma), 0.5% deoxycholate (Sigma), cOmplete EDTA-free protease inhibitor (Sigma)). The samples were vortexed for 10 minutes at 4°C and then centrifuged at 16,000 x g for 10 minutes at 4°C. Protein concentrations for each sample were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (1 µg/µL ) concentration.

Combined analysis:

Analysis ID	AN005270	AN005271
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:	CH003988
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:	MS005001
Analysis ID:	AN005270
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:	MS005002
Analysis ID:	AN005271
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