#METABOLOMICS WORKBENCH spinelli_lab_20240520_111432 DATATRACK_ID:4850 STUDY_ID:ST003218 ANALYSIS_ID:AN005276 PROJECT_ID:PR001988
VERSION             	1
CREATED_ON             	May 22, 2024, 7:58 pm
#PROJECT
PR:PROJECT_TITLE                 	Rhodoquinone is an Electron Carrier in the Mammalian Electron Transport Chain
PR:PROJECT_SUMMARY               	Ubiquinone (UQ), the only known electron carrier in the mammalian electron
PR:PROJECT_SUMMARY               	transport chain (ETC), delivers electrons to both oxygen (O2) and fumarate as
PR:PROJECT_SUMMARY               	terminal electron acceptors. As fumarate has a lower reduction potential than
PR:PROJECT_SUMMARY               	UQ, fumarate reduction is only thermodynamically favorable when ubiquinol, the
PR:PROJECT_SUMMARY               	reduced form of UQ, accumulates. Paradoxically, some tissues reduce fumarate
PR:PROJECT_SUMMARY               	without ubiquinol buildup, suggesting another mechanism enables fumarate
PR:PROJECT_SUMMARY               	reduction in mammals. Here, we identify rhodoquinone (RQ), a novel component of
PR:PROJECT_SUMMARY               	the mammalian ETC that carries electrons to fumarate, instead of O2, as the
PR:PROJECT_SUMMARY               	terminal electron acceptor. RQ, which is undetectable in cultured mammalian
PR:PROJECT_SUMMARY               	cells, is enriched in mitochondria from mouse and human tissues that catalyze
PR:PROJECT_SUMMARY               	high levels of fumarate reduction. UQ and RQ-directed ETC circuits support
PR:PROJECT_SUMMARY               	unique programs of mitochondrial function. Through expression of a bacterial
PR:PROJECT_SUMMARY               	enzyme that converts UQ into RQ and development of a novel RQ analog, we
PR:PROJECT_SUMMARY               	demonstrate that reprogramming the mammalian ETC from the UQ to RQ circuit
PR:PROJECT_SUMMARY               	renders cells highly resistant to hypoxia exposure in vitro and in vivo. Thus,
PR:PROJECT_SUMMARY               	we establish RQ as a fundamental component of the mammalian ETC and unveil that
PR:PROJECT_SUMMARY               	reprogramming the ETC to the RQ-circuit is a tractable strategy to ameliorate
PR:PROJECT_SUMMARY               	hypoxia-related conditions.
PR:INSTITUTE                     	UMass Chan Medical School
PR:LAST_NAME                     	UMass Chan
PR:FIRST_NAME                    	Spinelli Lab
PR:ADDRESS                       	55 Lake Avenue North, Worcester, Massachusetts, 01605, USA
PR:EMAIL                         	spinellilab@gmail.com
PR:PHONE                         	(508) 856-8989 ext. 68148
#STUDY
ST:STUDY_TITLE                   	Glutamine Tracing Assay in WT and SDHB KO 143B cells Treated with HKJS003
ST:STUDY_SUMMARY                 	WT and succinate dehydrogenase iron sulfur subunit B(SDHB) KO 143B cells were
ST:STUDY_SUMMARY                 	treated with a small molecule analog of Rhodoquinone, HKJS003. Glutamine tracing
ST:STUDY_SUMMARY                 	was used to determine the fumarate reduction and succinate oxidation of the
ST:STUDY_SUMMARY                 	treated cells. Other metabolites such as ATP,ADP,AMP,Glutathione, Oxidized
ST:STUDY_SUMMARY                 	glutathione, UTP, lactic acid, and pyruvic acid were also measured.
ST:INSTITUTE                     	UMass Chan Medical School
ST:LAST_NAME                     	UMass Chan
ST:FIRST_NAME                    	Spinelli Lab
ST:ADDRESS                       	55 Lake Avenue North, Worcester, Massachusetts, 01605, USA
ST:EMAIL                         	spinellilab@gmail.com
ST:PHONE                         	(508) 856-8989 ext. 68148
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#FACTORS
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	WT 1	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_01_20240402125032.raw
SUBJECT_SAMPLE_FACTORS           	-	WT 2	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_02.raw
SUBJECT_SAMPLE_FACTORS           	-	WT 3	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_03.raw
SUBJECT_SAMPLE_FACTORS           	-	WT 25nM 003 1	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_04.raw
SUBJECT_SAMPLE_FACTORS           	-	WT 25nM 003 2	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_05.raw
SUBJECT_SAMPLE_FACTORS           	-	WT 25nM 003 3	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_06.raw
SUBJECT_SAMPLE_FACTORS           	-	WT 100nM 003 1	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_07.raw
SUBJECT_SAMPLE_FACTORS           	-	WT 100nM 003 2	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_08.raw
SUBJECT_SAMPLE_FACTORS           	-	WT 100nM 003 3	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_09.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 1	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_10.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 2	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_11.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 3	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_12.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 25nM 003 1	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_13.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 25nM 003 2	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_14.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 25nM 003 3	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_15.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 100nM 003 1	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_16.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 100nM 003 2	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_17.raw
SUBJECT_SAMPLE_FACTORS           	-	SDHB KO 100nM 003 3	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003	Cell line=143B; RAW_FILE_NAME(File Name )=JV121_WT_vs_SDHBKO_003_18.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Media was aspirated from the plates and then the cells were washed with 1x PBS
CO:COLLECTION_SUMMARY            	twice. The plate was then transferred to dry ice and 500 µL of 80% HPLC-grade
CO:COLLECTION_SUMMARY            	methanol (Sigma) 20% HPLC-grade water (Sigma) was added to each well. The wells
CO:COLLECTION_SUMMARY            	were placed in a -80 freezer to incubate for 15 minutes. The plates are taken
CO:COLLECTION_SUMMARY            	out of the freezer one at a time and placed back on dry ice. The cells were then
CO:COLLECTION_SUMMARY            	scraped and transferred to a new tube.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	The 143B cell line were cultured in Dulbecco's Modified Eagle Medium (DMEM)
TR:TREATMENT_SUMMARY             	(ThermoFisher) supplemented with 10% Heat Inactivated Fetal Bovine Serum
TR:TREATMENT_SUMMARY             	(ThermoFisher) and 1% penicillin and streptomycin (ThermoFisher), and 100 µg/mL
TR:TREATMENT_SUMMARY             	uridine (Sigma). Cells were kept in a 37°C incubator (Baker Ruskinn) held at 5%
TR:TREATMENT_SUMMARY             	CO2 and 90% relative humidity.Cells were then supplemented with either DMSO or
TR:TREATMENT_SUMMARY             	HKJS003. For HKJS003, 20µL or 5µL of 100µM stock was added to 2mL of media
TR:TREATMENT_SUMMARY             	for a final concentration of 100nM or 25nM. For DMSO, 20µL of a diluted 1:1000
TR:TREATMENT_SUMMARY             	DMSO was added to 2mL of media. The cells were incubated for 5-7 of days to
TR:TREATMENT_SUMMARY             	proliferate and every 2 days the media was refreshed with the relevant
TR:TREATMENT_SUMMARY             	treatments.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Once cells were collected into Eppendorf tubes, each well was washed with an
SP:SAMPLEPREP_SUMMARY            	additional 300 µL of 80% HPLC-grade methanol (Sigma) 20% HPLC-grade water
SP:SAMPLEPREP_SUMMARY            	(Sigma) and collected into the same tube as the initial lysis. The samples were
SP:SAMPLEPREP_SUMMARY            	then vortexed at 4° C for 10 minutes and centrifuged at 21,300 x g for 10
SP:SAMPLEPREP_SUMMARY            	minutes at 4° C. Supernatants were transferred to a new tube and dried down in
SP:SAMPLEPREP_SUMMARY            	a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a
SP:SAMPLEPREP_SUMMARY            	CentriVap-105 Cold Trap (Labconco). After being dried down, pellets were stored
SP:SAMPLEPREP_SUMMARY            	in a -20° C freezer until ready to run on the polar LC-MS method.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	20 min, 80% to 20% B; 0.5 min, 20% to 80% B; 7.5min, 80% B
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
CH:SOLVENT_A                     	100% water; 0.1% ammonium hydroxide; 20mM ammonium carbonate
CH:SOLVENT_B                     	100% acetonitrile
CH:FLOW_GRADIENT                 	Gradient starts at 80% B. Subsequently, 80% to 20% B linearly in 20min; 20% to
CH:FLOW_GRADIENT                 	80% B in 0.5min; 80% B for 7.5min
CH:FLOW_RATE                     	0.15ml/min
CH:COLUMN_TEMPERATURE            	25
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Plus Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The mass spectrometer was set to full scan (70-1000 m/z), with the spray voltage
MS:MS_COMMENTS                   	set to 4.0 kV, heated capillary to 350°C, and the HESI probe at 30 °C. The
MS:MS_COMMENTS                   	sheath gas flow was set at 10 units, auxiliary gas at 1 units, and sweep gas
MS:MS_COMMENTS                   	flow at 1 unit. The resolution of scan was set to 70,000, AGC target to 1x106,
MS:MS_COMMENTS                   	and maximum injection time at 20 msec. Data acquired by Thermo Fisher's Xcalibur
MS:MS_COMMENTS                   	software and analyzed by their Tracefinder software. The raw files provided
MS:MS_COMMENTS                   	contain data from both positive and negative ion mode.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area
MS_METABOLITE_DATA_START
Samples	WT 1	WT 2	WT 3	WT 25nM 003 1	WT 25nM 003 2	WT 25nM 003 3	WT 100nM 003 1	WT 100nM 003 2	WT 100nM 003 3	SDHB KO 1	SDHB KO 2	SDHB KO 3	SDHB KO 25nM 003 1	SDHB KO 25nM 003 2	SDHB KO 25nM 003 3	SDHB KO 100nM 003 1	SDHB KO 100nM 003 2	SDHB KO 100nM 003 3
Factors	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:DMSO	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:25nM HKJS003	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003	Sample source:Bone sarcoma cells | Genotype:WT | Treatment:100nM HKJS003	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:DMSO	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:25nM HKJS003	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003	Sample source:Bone sarcoma cells | Genotype:SDHB KO | Treatment:100nM HKJS003
Glutathione	292698418	328802279.1	347059528.3	373996007.1	373796867.5	294696223.6	264256565.6	369591153.2	356520964.6	489143105.5	398313414.5	464985477.2	386502434	392226487.6	513853489.9	228270296.2	393312164.7	378939255.4
Oxidized glutathione	6793757.65	5875048.016	6368457.519	8962317.544	6372007.147	3304381.612	2509261.029	5414605.288	5848973.377	11081976.32	7151053.296	7806179.079	10722281.68	8854580.138	11768190.19	3508012.008	6988012.485	7449209.352
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Retention index	Quantified m/z	PubChem ID	KEGG ID
Glutathione	9.5	308.09108	124886	C00051
Oxidized glutathione	10.5	613.15924	65359	C00127
METABOLITES_END
#END