Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA046973

Local Sample ID:	ms5959-18
Subject ID:	SU000875
Subject Type:	Mouse
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammal

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Subject:

Subject ID:	SU000875
Subject Type:	Mouse
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
ms5959-18	SA046973	FL009290	P60d	time pt
ms5959-18	SA046973	FL009290	PAR1-/-	grouping

Collection:

Collection ID:	CO000869
Collection Summary:	The samples submitted are purified myelin preparations from the postnatal day 21 or 90 mouse spinal cord (SC). There are two projects but these can be run together since the down stream assays are identical. There are 9 samples total in Project 1, n=3 for each genotype (WT, PAR1-/- or PAR2-/-). There are 12 samples total in Project 2, n=3 for K6+/+ or K6-/- at either P21 or P90. We are submitting these samples for analysis of 1) free fatty acid panel; 2) free fatty acid composition of lipids; 3) cholesterol concentration (free and bound); 4) Ceramides, including galactosyl and glucosyl; 5) sphingomyelin. A total of approximately 130 to 150 ul for each sample is submitted with the protein concentrations already measured by BCA assay and indicated below. Concentrations are provided in ug/ul and the total volume is also indicated. One tube for each sample is submitted but this can be shared across the assays.
Sample Type:	Spinal cord

Treatment:

Treatment ID:	TR000889
Treatment Summary:	A 3g Clip produces moderate SCI including demyelination and clinical impairment and we recently published a detailed methodology. At 1 week after injury, the 3g injured mice are expected to have an average Basso Mouse Scale score (BMS)=5 on a 9 point scale such that they have frequent plantar stepping with no or some coordination. This level of impairment was chosen to provide a sufficient window to observe recovery.

Treatment ID:

TR000889

Treatment Summary:

A 3g Clip produces moderate SCI including demyelination and clinical impairment and we recently published a detailed methodology. At 1 week after injury, the 3g injured mice are expected to have an average Basso Mouse Scale score (BMS)=5 on a 9 point scale such that they have frequent plantar stepping with no or some coordination. This level of impairment was chosen to provide a sufficient window to observe recovery.

Sample Preparation:

Sampleprep ID:	SP000882
Sampleprep Summary:	NEFA of mouse spinal cord Lipids will be quantified in myelin isolated in high yield and purity by subcellular fractionation from the lumbosacral spinal cord. While there are no absolutely ‘myelin-specific’ lipids, galactocerebroside is the most typical of myelin in the adult nervous system being directly proportional to the amount of myelin. Sulfatide is another galactolipid enriched in myelin. Together with cholesterol, these form 78% of the total amount of lipid in the myelin membrane and each will be quantified using LC/MS/MS. A highly sensitive assay for galactocerebroside was recently established by the Mayo Metabolomics Core and can be implemented immediately. The LC/MS/MS panel for free fatty acids, including the very long chain fatty acids found in myelin is also routinely performed by the Core. Cholesterol will be quantified using an NMR-based approach by the Mayo Dept. of Laboratory Medicine Clinical Core. Additionally, we have a plan in place with the Metabolomics Core to develop LC/MS/MS assays for sulfatide and sphingomyelin during the Pilot proposal. Having quantitative assays for each of these key myelin lipids will facilitate our goal to comprehensively profile myelin lipid metabolism and will form foundational assays for a future NIH grant focused on myelin metabolism.

Sampleprep ID:

SP000882

Sampleprep Summary:

NEFA of mouse spinal cord Lipids will be quantified in myelin isolated in high yield and purity by subcellular fractionation from the lumbosacral spinal cord. While there are no absolutely ‘myelin-specific’ lipids, galactocerebroside is the most typical of myelin in the adult nervous system being directly proportional to the amount of myelin. Sulfatide is another galactolipid enriched in myelin. Together with cholesterol, these form 78% of the total amount of lipid in the myelin membrane and each will be quantified using LC/MS/MS. A highly sensitive assay for galactocerebroside was recently established by the Mayo Metabolomics Core and can be implemented immediately. The LC/MS/MS panel for free fatty acids, including the very long chain fatty acids found in myelin is also routinely performed by the Core. Cholesterol will be quantified using an NMR-based approach by the Mayo Dept. of Laboratory Medicine Clinical Core. Additionally, we have a plan in place with the Metabolomics Core to develop LC/MS/MS assays for sulfatide and sphingomyelin during the Pilot proposal. Having quantitative assays for each of these key myelin lipids will facilitate our goal to comprehensively profile myelin lipid metabolism and will form foundational assays for a future NIH grant focused on myelin metabolism.

Combined analysis:

Analysis ID	AN001372
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity
Column	Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Agilent 6460 QQQ
Ion Mode	NEGATIVE
Units	ng/ug of protein

Chromatography:

Chromatography ID:	CH000957
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001264
Analysis ID:	AN001372
Instrument Name:	Agilent 6460 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	NEGATIVE