Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA136773

Local Sample ID:	Sample4-NoISD
Subject ID:	SU001688
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU001688
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Sample4-NoISD	SA136773	FL017502	Skeletal muscle	Treatment

Collection:

Collection ID:	CO001681
Collection Summary:	5 skeletal muscle and 5 sarcoma samples. Tissue polar metabolites were extracted into 80% methanol/water.
Sample Type:	Blood;Sarcoma;Skeletal muscle

Treatment:

Treatment ID:	TR001701
Treatment Summary:	The mouse model of soft-tissue sarcoma was generated on a mixed background (129/SvJae and C57BL/6) using a combination of alleles that were previously described: Pax7CreER-T2 20, p53FL/FL 21, LSL-NrasG12D 22 and ROSA26mTmG Primary mouse soft tissue sarcomas were generated in the mouse hind limb as previously described 19 by intramuscular (IM) injection of (Z)-4-hydroxytamoxifen (4-OHT). 4-OHT was dissolved in 100% DMSO at a concentration of 10 mg/ml and 50 ul of the solution was injected into the gastrocnemius muscle.

Treatment ID:

TR001701

Treatment Summary:

The mouse model of soft-tissue sarcoma was generated on a mixed background (129/SvJae and C57BL/6) using a combination of alleles that were previously described: Pax7CreER-T2 20, p53FL/FL 21, LSL-NrasG12D 22 and ROSA26mTmG Primary mouse soft tissue sarcomas were generated in the mouse hind limb as previously described 19 by intramuscular (IM) injection of (Z)-4-hydroxytamoxifen (4-OHT). 4-OHT was dissolved in 100% DMSO at a concentration of 10 mg/ml and 50 ul of the solution was injected into the gastrocnemius muscle.

Sample Preparation:

Sampleprep ID:	SP001694
Sampleprep Summary:	The tumor sample was first homogenized in liquid nitrogen and then 5 to 10 mg was weighed in a new Eppendorf tube. Ice cold extraction solvent (250 ul 80% MeOH/water) was added to the tissue sample, and a pellet mixer was used to further break down the tissue chunk and form an even suspension, followed by the addition of 250 ul to rinse the pellet mixer. After incubation on ice for an additional 10 min, the tissue extract was centrifuged with a speed of 20,000 g at 4 °C for 10 min. The dry pellets were reconstituted into 30 ul (per 3 mg tissue) sample solvent (water:methanol: acetonitrile, 2:1:1, v/v), and 3 ul was injected to LC-HRMS. 5 ul mouse plasma was mixed with 5 ul water, and 40 ul ice cold methanol was added. After vortex for 1 min, the mixture was centrifuged with a speed of 20,000 g at 4 °C for 10 min, and 3 l was injected to LC-HRMS.

Sampleprep ID:

SP001694

Sampleprep Summary:

The tumor sample was first homogenized in liquid nitrogen and then 5 to 10 mg was weighed in a new Eppendorf tube. Ice cold extraction solvent (250 ul 80% MeOH/water) was added to the tissue sample, and a pellet mixer was used to further break down the tissue chunk and form an even suspension, followed by the addition of 250 ul to rinse the pellet mixer. After incubation on ice for an additional 10 min, the tissue extract was centrifuged with a speed of 20,000 g at 4 °C for 10 min. The dry pellets were reconstituted into 30 ul (per 3 mg tissue) sample solvent (water:methanol: acetonitrile, 2:1:1, v/v), and 3 ul was injected to LC-HRMS. 5 ul mouse plasma was mixed with 5 ul water, and 40 ul ice cold methanol was added. After vortex for 1 min, the mixture was centrifuged with a speed of 20,000 g at 4 °C for 10 min, and 3 l was injected to LC-HRMS.

Combined analysis:

Analysis ID	AN002645	AN002646
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000
Column	Waters Xbridge amide (100 x 2.1mm,3.5um)	Waters Xbridge amide (100 x 2.1mm,3.5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap	Thermo Exactive Plus Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH001954
Chromatography Summary:	HILIC method is for general metabolomics analysis.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters Xbridge amide (100 x 2.1mm,3.5um)
Chromatography Type:	HILIC

MS:

MS ID:	MS002457
Analysis ID:	AN002645
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	It was operated in the full-scan mode with the resolution set at 70 000 (at m/z 200).LC-MS data were analyzed using Sieve 2.0 (Thermo Scientific), and the integrated area under metabolite peak was used to compare the relative abundance of each metabolite in different samples in the same batch.
Ion Mode:	POSITIVE

MS ID:	MS002458
Analysis ID:	AN002646
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	It was operated in the full-scan mode with the resolution set at 70 000 (at m/z 200).LC-MS data were analyzed using Sieve 2.0 (Thermo Scientific), and the integrated area under metabolite peak was used to compare the relative abundance of each metabolite in different samples in the same batch.
Ion Mode:	NEGATIVE