Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA308603

Local Sample ID:	Neg_WT_4
Subject ID:	SU002962
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Mammals

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Subject:

Subject ID:	SU002962
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Mammals

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Neg_WT_4	SA308603	FL037160	Wild-type	Genotype

Collection:

Collection ID:	CO002955
Collection Summary:	Primary mus musculus cells
Sample Type:	Osteoclast

Treatment:

Treatment ID:	TR002971
Treatment Summary:	All in vitro experiments were performed at least 3 times. Primary bone marrow macrophages (BMMs) were prepared as described. Marrow was extracted from femora and tibiae of 6- to 8-week-old mice with α minimum essential medium (α-MEM) and cultured in α-MEM containing 10% inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (α-10 medium) with 1:10 of mMCSF producing cell line, CMG 14-12 condition media on petri-plastic dishes. Cells were incubated at 37°C in 5% CO2 for 3 days and then washed with phosphate-buffered saline (PBS) and lifted with 1X trypsin/EDTA in PBS. A total of 1.2 × 104 BMMs were cultured in 500 μL α-MEM containing 10% heat-inactivated fetal bovine serum with glutathione-S transferase–RANKL and 30 ng/mL of mouse recombinant macrophage colony-stimulating factor (M-CSF) in 48-well tissue culture plates, some containing sterile bovine bone slices.

Treatment ID:

TR002971

Treatment Summary:

All in vitro experiments were performed at least 3 times. Primary bone marrow macrophages (BMMs) were prepared as described. Marrow was extracted from femora and tibiae of 6- to 8-week-old mice with α minimum essential medium (α-MEM) and cultured in α-MEM containing 10% inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (α-10 medium) with 1:10 of mMCSF producing cell line, CMG 14-12 condition media on petri-plastic dishes. Cells were incubated at 37°C in 5% CO2 for 3 days and then washed with phosphate-buffered saline (PBS) and lifted with 1X trypsin/EDTA in PBS. A total of 1.2 × 104 BMMs were cultured in 500 μL α-MEM containing 10% heat-inactivated fetal bovine serum with glutathione-S transferase–RANKL and 30 ng/mL of mouse recombinant macrophage colony-stimulating factor (M-CSF) in 48-well tissue culture plates, some containing sterile bovine bone slices.

Sample Preparation:

Sampleprep ID:	SP002968
Sampleprep Summary:	Cells were quenched with cold LC/MS-grade methanol, then scraped and transferred to Eppendorf tubes. Samples were dried in a SpeedVac. The samples were then reconstituted in 1 mL of cold methanol:acetonitrile:water (2:2:1) and subjected to three cycles of vortexing, freezing in liquid nitrogen, and 10 min of sonication at 25 °C. Samples were stored at −20 °C overnight and then centrifuged for 10 min at 14,000×g and 4 °C. Supernatants were transferred to new tubes and dried by a SpeedVac. The protein abundance of each sample was determined by using BCA. A quantity of 1 μl of acetonitrile:water (2:1) per each 2.5 μg of protein was used. Samples were subjected to two cycles of vortexing and 10 min of sonication at 25 °C. Next, samples were centrifuged for 10 min at 14,000×g and 4 °C, transferred supernatant to LC vials, and stored at −80 °C until MS analysis

Sampleprep ID:

SP002968

Sampleprep Summary:

Cells were quenched with cold LC/MS-grade methanol, then scraped and transferred to Eppendorf tubes. Samples were dried in a SpeedVac. The samples were then reconstituted in 1 mL of cold methanol:acetonitrile:water (2:2:1) and subjected to three cycles of vortexing, freezing in liquid nitrogen, and 10 min of sonication at 25 °C. Samples were stored at −20 °C overnight and then centrifuged for 10 min at 14,000×g and 4 °C. Supernatants were transferred to new tubes and dried by a SpeedVac. The protein abundance of each sample was determined by using BCA. A quantity of 1 μl of acetonitrile:water (2:1) per each 2.5 μg of protein was used. Samples were subjected to two cycles of vortexing and 10 min of sonication at 25 °C. Next, samples were centrifuged for 10 min at 14,000×g and 4 °C, transferred supernatant to LC vials, and stored at −80 °C until MS analysis

Combined analysis:

Analysis ID	AN004668	AN004669
Chromatography ID	CH003514	CH003514
MS ID	MS004415	MS004416
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish Flex UHPLC Systems	Thermo Vanquish Flex UHPLC Systems
Column	HILICON iHILIC-(P) Classic (100 x 2.1mm,5um)	HILICON iHILIC-(P) Classic (100 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Orbitrap ID-X Tribrid	Thermo Orbitrap ID-X Tribrid
Ion Mode	NEGATIVE	POSITIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH003514
Instrument Name:	Thermo Vanquish Flex UHPLC Systems
Column Name:	HILICON iHILIC-(P) Classic (100 x 2.1mm,5um)
Column Temperature:	45
Flow Gradient:	0–1 min: 90% B, 1–12 min: 90-35% B, 12–12.5 min: 35-25% B, 12.5–14.5 min: 25% B
Flow Rate:	250 uL/min
Solvent A:	20 mM ammonium bicarbonate, 0.1% ammonium hydroxideand 2.5 μM medronic acid in water:acetonitrile (95:5)
Solvent B:	acetonitrile:water (95:5)
Chromatography Type:	HILIC

MS:

MS ID:	MS004415
Analysis ID:	AN004668
Instrument Name:	Thermo Orbitrap ID-X Tribrid
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data were collected with the following MS source settings: spray voltage, -2.8 kV; sheath gas, 50; auxiliary gas, 10; sweep gas, 1; ion transfer tube temperature, 300°C; vaporizer temperature, 200°C; mass range, 67 – 1000 Da; resolution, 120,000; maximum injection time, 200 ms; isolation window, 1.5 Da. XCMS and Skyline software were used for data processing
Ion Mode:	NEGATIVE

MS ID:	MS004416
Analysis ID:	AN004669
Instrument Name:	Thermo Orbitrap ID-X Tribrid
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data were collected with the following MS source settings: spray voltage, 3.5 kV; sheath gas, 50; auxiliary gas, 10; sweep gas, 1; ion transfer tube temperature, 300°C; vaporizer temperature, 200°C; mass range, 67 – 1000 Da; resolution, 120,000; maximum injection time, 200 ms; isolation window, 1.5 Da. XCMS and Skyline software were used for data processing
Ion Mode:	POSITIVE