#METABOLOMICS WORKBENCH kevin.cho@wustl.edu_20230826_084448 DATATRACK_ID:4250 STUDY_ID:ST002850 ANALYSIS_ID:AN004668 PROJECT_ID:PR001784
VERSION             	1
CREATED_ON             	September 8, 2023, 9:11 am
#PROJECT
PR:PROJECT_TITLE                 	Bap1 Promotes Osteoclast Function by Metabolic Reprogramming
PR:PROJECT_TYPE                  	Untargeted Metabolomics
PR:PROJECT_SUMMARY               	Treatment of osteoporosis most commonly diminishes osteoclast number which
PR:PROJECT_SUMMARY               	suppresses bone formation thus compromising fracture prevention. Bone formation
PR:PROJECT_SUMMARY               	is not suppressed, however, when bone degradation is reduced by retarding
PR:PROJECT_SUMMARY               	osteoclast functional resorptive capacity, rather than differentiation. We find
PR:PROJECT_SUMMARY               	deletion of deubiquitinase, BRCA1-associated protein 1 (Bap1), in myeloid cells
PR:PROJECT_SUMMARY               	(Bap1∆LysM), arrests osteoclast function but not formation. Bap1∆LysM
PR:PROJECT_SUMMARY               	osteoclasts fail to organize their cytoskeleton which is essential for bone
PR:PROJECT_SUMMARY               	degradation. Consequently, bone mass increases in the mutant mice. We find the
PR:PROJECT_SUMMARY               	deubiquitinase activity of Bap1 regulates osteoclast function by metabolic
PR:PROJECT_SUMMARY               	reprogramming. Bap1 deficient osteoclast lineage cells upregulate the cystine
PR:PROJECT_SUMMARY               	transporter, Slc7a11, by enhanced H2Aub occupancy of its promoter. SLC7A11
PR:PROJECT_SUMMARY               	regulates cellular ROS levels and redirects the mitochondrial metabolites away
PR:PROJECT_SUMMARY               	from the TCA cycle, both of which are necessary for osteoclast function. Thus in
PR:PROJECT_SUMMARY               	osteoclasts, Bap1 appears to regulate epigenetic-metabolic axis and is a
PR:PROJECT_SUMMARY               	potential target to reduce bone degradation while maintaining osteogenesis in
PR:PROJECT_SUMMARY               	osteoporotic patients.
PR:INSTITUTE                     	Washington University in St. Louis
PR:DEPARTMENT                    	Pathology and Immunology, Medicine, Chemistry
PR:LABORATORY                    	Teitelbaum and Patti Laboratories
PR:LAST_NAME                     	Cho
PR:FIRST_NAME                    	Kevin
PR:ADDRESS                       	1 Brookings Drive, Campus Box 1134, St. Louis, MO, 63130, USA
PR:EMAIL                         	kevin.cho@wustl.edu
PR:PHONE                         	314-935-8813
#STUDY
ST:STUDY_TITLE                   	Bap1 Promotes Osteoclast Function by Metabolic Reprogramming
ST:STUDY_TYPE                    	Untargeted Metabolomics
ST:STUDY_SUMMARY                 	Treatment of osteoporosis most commonly diminishes osteoclast number which
ST:STUDY_SUMMARY                 	suppresses bone formation thus compromising fracture prevention. Bone formation
ST:STUDY_SUMMARY                 	is not suppressed, however, when bone degradation is reduced by retarding
ST:STUDY_SUMMARY                 	osteoclast functional resorptive capacity, rather than differentiation. We find
ST:STUDY_SUMMARY                 	deletion of deubiquitinase, BRCA1-associated protein 1 (Bap1), in myeloid cells
ST:STUDY_SUMMARY                 	(Bap1∆LysM), arrests osteoclast function but not formation. Bap1∆LysM
ST:STUDY_SUMMARY                 	osteoclasts fail to organize their cytoskeleton which is essential for bone
ST:STUDY_SUMMARY                 	degradation. Consequently, bone mass increases in the mutant mice. We find the
ST:STUDY_SUMMARY                 	deubiquitinase activity of Bap1 regulates osteoclast function by metabolic
ST:STUDY_SUMMARY                 	reprogramming. Bap1 deficient osteoclast lineage cells upregulate the cystine
ST:STUDY_SUMMARY                 	transporter, Slc7a11, by enhanced H2Aub occupancy of its promoter. SLC7A11
ST:STUDY_SUMMARY                 	regulates cellular ROS levels and redirects the mitochondrial metabolites away
ST:STUDY_SUMMARY                 	from the TCA cycle, both of which are necessary for osteoclast function. Thus in
ST:STUDY_SUMMARY                 	osteoclasts, Bap1 appears to regulate epigenetic-metabolic axis and is a
ST:STUDY_SUMMARY                 	potential target to reduce bone degradation while maintaining osteogenesis in
ST:STUDY_SUMMARY                 	osteoporotic patients.
ST:INSTITUTE                     	Washington University in St. Louis
ST:DEPARTMENT                    	Pathology and Immunology, Medicine, Chemistry
ST:LABORATORY                    	Teitelbaum and Patti Laboratories
ST:LAST_NAME                     	Cho
ST:FIRST_NAME                    	Kevin
ST:ADDRESS                       	1 Brookings Drive, Campus Box 1134, St. Louis, MO, 63130, USA
ST:EMAIL                         	kevin.cho@wustl.edu
ST:PHONE                         	314-935-8813
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	-	Pos_WT_1	Genotype:Wild-type	Polarity=Positive; RAW_FILE_NAME=Pos_WT_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_WT_2	Genotype:Wild-type	Polarity=Positive; RAW_FILE_NAME=Pos_WT_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_WT_3	Genotype:Wild-type	Polarity=Positive; RAW_FILE_NAME=Pos_WT_3.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_WT_4	Genotype:Wild-type	Polarity=Positive; RAW_FILE_NAME=Pos_WT_4.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_WT_5	Genotype:Wild-type	Polarity=Positive; RAW_FILE_NAME=Pos_WT_5.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_KO_1	Genotype:Knockout	Polarity=Positive; RAW_FILE_NAME=Pos_KO_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_KO_2	Genotype:Knockout	Polarity=Positive; RAW_FILE_NAME=Pos_KO_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_KO_3	Genotype:Knockout	Polarity=Positive; RAW_FILE_NAME=Pos_KO_3.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_KO_4	Genotype:Knockout	Polarity=Positive; RAW_FILE_NAME=Pos_KO_4.mzML
SUBJECT_SAMPLE_FACTORS           	-	Pos_KO_5	Genotype:Knockout	Polarity=Positive; RAW_FILE_NAME=Pos_KO_5.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_WT_1	Genotype:Wild-type	Polarity=Negative; RAW_FILE_NAME=Neg_WT_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_WT_2	Genotype:Wild-type	Polarity=Negative; RAW_FILE_NAME=Neg_WT_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_WT_3	Genotype:Wild-type	Polarity=Negative; RAW_FILE_NAME=Neg_WT_3.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_WT_4	Genotype:Wild-type	Polarity=Negative; RAW_FILE_NAME=Neg_WT_4.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_WT_5	Genotype:Wild-type	Polarity=Negative; RAW_FILE_NAME=Neg_WT_5.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_KO_1	Genotype:Knockout	Polarity=Negative; RAW_FILE_NAME=Neg_KO_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_KO_2	Genotype:Knockout	Polarity=Negative; RAW_FILE_NAME=Neg_KO_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_KO_3	Genotype:Knockout	Polarity=Negative; RAW_FILE_NAME=Neg_KO_3.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_KO_4	Genotype:Knockout	Polarity=Negative; RAW_FILE_NAME=Neg_KO_4.mzML
SUBJECT_SAMPLE_FACTORS           	-	Neg_KO_5	Genotype:Knockout	Polarity=Negative; RAW_FILE_NAME=Neg_KO_5.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Primary mus musculus cells
CO:SAMPLE_TYPE                   	Osteoclast
#TREATMENT
TR:TREATMENT_SUMMARY             	All in vitro experiments were performed at least 3 times. Primary bone marrow
TR:TREATMENT_SUMMARY             	macrophages (BMMs) were prepared as described with slight modification. Marrow
TR:TREATMENT_SUMMARY             	was extracted from femora and tibiae of 6- to 8-week-old mice with α minimum
TR:TREATMENT_SUMMARY             	essential medium (α-MEM) and cultured in α-MEM containing 10% inactivated
TR:TREATMENT_SUMMARY             	fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (α-10
TR:TREATMENT_SUMMARY             	medium) with 1:10 of mMCSF producing cell line, CMG 14-12 condition media on
TR:TREATMENT_SUMMARY             	petri-plastic dishes. Cells were incubated at 37°C in 5% CO2 for 3 days and
TR:TREATMENT_SUMMARY             	then washed with phosphate-buffered saline (PBS) and lifted with 1X trypsin/EDTA
TR:TREATMENT_SUMMARY             	in PBS. A total of 1.2 × 104 BMMs were cultured in 500 μL α-MEM containing
TR:TREATMENT_SUMMARY             	10% heat-inactivated fetal bovine serum with glutathione-S transferase–RANKL
TR:TREATMENT_SUMMARY             	and 30 ng/mL of mouse recombinant macrophage colony-stimulating factor (M-CSF)
TR:TREATMENT_SUMMARY             	in 48-well tissue culture plates, some containing sterile bovine bone slices.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Cells were quenched with cold LC/MS-grade methanol, then scraped and transferred
SP:SAMPLEPREP_SUMMARY            	to Eppendorf tubes. Samples were dried in a SpeedVac. The samples were then
SP:SAMPLEPREP_SUMMARY            	reconstituted in 1 mL of cold methanol:acetonitrile:water (2:2:1) and subjected
SP:SAMPLEPREP_SUMMARY            	to three cycles of vortexing, freezing in liquid nitrogen, and 10 min of
SP:SAMPLEPREP_SUMMARY            	sonication at 25 °C. Samples were stored at −20 °C overnight and then
SP:SAMPLEPREP_SUMMARY            	centrifuged for 10 min at 14,000×g and 4 °C. Supernatants were transferred to
SP:SAMPLEPREP_SUMMARY            	new tubes and dried by a SpeedVac. The protein abundance of each sample was
SP:SAMPLEPREP_SUMMARY            	determined by using BCA. A quantity of 1 μl of acetonitrile:water (2:1) per
SP:SAMPLEPREP_SUMMARY            	each 2.5 μg of protein was used. Samples were subjected to two cycles of
SP:SAMPLEPREP_SUMMARY            	vortexing and 10 min of sonication at 25 °C. Next, samples were centrifuged for
SP:SAMPLEPREP_SUMMARY            	10 min at 14,000×g and 4 °C, transferred supernatant to LC vials, and stored
SP:SAMPLEPREP_SUMMARY            	at −80 °C until MS analysis
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Thermo Vanquish Flex UHPLC Systems
CH:COLUMN_NAME                   	HILICON iHILIC-(P) Classic (100 x 2.1mm,5um)
CH:SOLVENT_A                     	20 mM ammonium bicarbonate, 0.1% ammonium hydroxideand 2.5 μM medronic acid in
CH:SOLVENT_A                     	water:acetonitrile (95:5)
CH:SOLVENT_B                     	acetonitrile:water (95:5)
CH:FLOW_GRADIENT                 	0–1 min: 90% B, 1–12 min: 90-35% B, 12–12.5 min: 35-25% B, 12.5–14.5
CH:FLOW_GRADIENT                 	min: 25% B
CH:FLOW_RATE                     	250 uL/min
CH:COLUMN_TEMPERATURE            	45
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap ID-X tribrid
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Data were collected with the following MS source settings: spray voltage, -2.8
MS:MS_COMMENTS                   	kV; sheath gas, 50; auxiliary gas, 10; sweep gas, 1; ion transfer tube
MS:MS_COMMENTS                   	temperature, 300°C; vaporizer temperature, 200°C; mass range, 67 – 1000 Da;
MS:MS_COMMENTS                   	resolution, 120,000; maximum injection time, 200 ms; isolation window, 1.5 Da.
MS:MS_COMMENTS                   	XCMS and Skyline software were used for data processing
MS:MS_RESULTS_FILE               	ST002850_AN004668_Results.txt	UNITS:peak area	Has m/z:Yes	Has RT:Yes	RT units:Seconds
#END