Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA322026

Local Sample ID:	212-3748_brain
Subject ID:	SU003071
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU003071
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
212-3748_brain	SA322026	FL038426	Brain	Tissue
212-3748_brain	SA322026	FL038426	+	Genotype
212-3748_brain	SA322026	FL038426	Week 12	Timepoint
212-3748_brain	SA322026	FL038426	Control	Treatment

Collection:

Collection ID:	CO003064
Collection Summary:	Euthanasia was performed by carbon dioxide inhalation and cervical dislocation, and serum, brain, and spinal cord tissues were collected immediately, frozen on dry ice, and stored at -80 °C until processing.
Sample Type:	Brain; Spinal cord; Serum

Treatment:

Treatment ID:	TR003080
Treatment Summary:	Plp1-iCKO-Myrf mice were randomly assigned into the control or Sob-AM2 treatment groups. Both Cre negative mice, which do not lose Myrf and undergo demyelination, and Cre positive mice, which do undergo demyelination, were used in all experiments. The control group received control chow (Envigo Teklad 2016 diet) and the Sob-AM2 treatment group received chow containing 420 µg/kg chow of Sob-AM2 (nominal daily dose of 84 µg/kg body weight) starting 2 weeks after tamoxifen injections. Mice on control chow were euthanized at 4 timepoints: 6, 12, 18, and 24 weeks post-tamoxifen injections. Mice treated with Sob-AM2 were euthanized at 12 and 18 weeks post-tamoxifen.

Treatment ID:

TR003080

Treatment Summary:

Plp1-iCKO-Myrf mice were randomly assigned into the control or Sob-AM2 treatment groups. Both Cre negative mice, which do not lose Myrf and undergo demyelination, and Cre positive mice, which do undergo demyelination, were used in all experiments. The control group received control chow (Envigo Teklad 2016 diet) and the Sob-AM2 treatment group received chow containing 420 µg/kg chow of Sob-AM2 (nominal daily dose of 84 µg/kg body weight) starting 2 weeks after tamoxifen injections. Mice on control chow were euthanized at 4 timepoints: 6, 12, 18, and 24 weeks post-tamoxifen injections. Mice treated with Sob-AM2 were euthanized at 12 and 18 weeks post-tamoxifen.

Sample Preparation:

Sampleprep ID:	SP003077
Sampleprep Summary:	A modified Bligh-Dyer protocol was used to extract lipids from the brain and spinal cord tissue. Brain homogenates (either 300 mg/ml or 50 mg/ml) were prepared with ice cold water using a Bead Mill homogenizer (Bead Ruptor Elite, Omni international, USA). Spinal cord homogenates were prepared at 65 mg/ml in cold water. Immediately after homogenization, the brain homogenates were diluted with cold water (150 µL of 300 mg/ml brain homogenate was mixed with 800 µl of cold water. For the 50 mg/ml brain homogenates, 1 mL of homogenate was used directly. Spinal cord homogenates were diluted 10-fold (100 μl of the spinal cord homogenates with 900 μl of cold water). For all samples, 10 μl of the diluted homogenate was removed and stored at -80°C for protein quantification using a BCA assay. The diluted tissue homogenates were combined with a mixture of chloroform (containing 0.01% butylated hydroxytoluene, BHT): methanol: water (3:2:1) in glass tubes. After shaking and vortexing thoroughly, the mixture was centrifuged (Sorvall ST 40R Centrifuge, Thermo Fisher Scientific) at 1300 rpm for 10 minutes. The lower layer was carefully removed and saved in a glass tube. The remaining top layer was further extracted twice with 1.25 ml chloroform with 0.01% BHT; the lower layers were carefully removed and combined. The combined lower layer was then washed with 300 μl of 1 M KCl followed by 300 μl of water and vacuum dried completely (Savant SpeedVac SPD130DLX vacuum concentrator, Thermo Fisher Scientific, USA) to obtain the dried lipid extract. Serum (3 μl) was added directly to a vial containing 1.2 mL of chloroform: methanol: 300 mM ammonium acetate in water (300:665:35). The contents were mixed thoroughly, and the vials were centrifuged for 5 min at low speed in a clinical centrifuge to pellet proteins before submitting samples to the mass spectrometer.

Sampleprep ID:

SP003077

Sampleprep Summary:

A modified Bligh-Dyer protocol was used to extract lipids from the brain and spinal cord tissue. Brain homogenates (either 300 mg/ml or 50 mg/ml) were prepared with ice cold water using a Bead Mill homogenizer (Bead Ruptor Elite, Omni international, USA). Spinal cord homogenates were prepared at 65 mg/ml in cold water. Immediately after homogenization, the brain homogenates were diluted with cold water (150 µL of 300 mg/ml brain homogenate was mixed with 800 µl of cold water. For the 50 mg/ml brain homogenates, 1 mL of homogenate was used directly. Spinal cord homogenates were diluted 10-fold (100 μl of the spinal cord homogenates with 900 μl of cold water). For all samples, 10 μl of the diluted homogenate was removed and stored at -80°C for protein quantification using a BCA assay. The diluted tissue homogenates were combined with a mixture of chloroform (containing 0.01% butylated hydroxytoluene, BHT): methanol: water (3:2:1) in glass tubes. After shaking and vortexing thoroughly, the mixture was centrifuged (Sorvall ST 40R Centrifuge, Thermo Fisher Scientific) at 1300 rpm for 10 minutes. The lower layer was carefully removed and saved in a glass tube. The remaining top layer was further extracted twice with 1.25 ml chloroform with 0.01% BHT; the lower layers were carefully removed and combined. The combined lower layer was then washed with 300 μl of 1 M KCl followed by 300 μl of water and vacuum dried completely (Savant SpeedVac SPD130DLX vacuum concentrator, Thermo Fisher Scientific, USA) to obtain the dried lipid extract. Serum (3 μl) was added directly to a vial containing 1.2 mL of chloroform: methanol: 300 mM ammonium acetate in water (300:665:35). The contents were mixed thoroughly, and the vials were centrifuged for 5 min at low speed in a clinical centrifuge to pellet proteins before submitting samples to the mass spectrometer.

Combined analysis:

Analysis ID	AN004858
Analysis type	MS
Chromatography type	None (Direct infusion)
Chromatography system	Sciex 4000 QTrap
Column	none
MS Type	ESI
MS instrument type	QTRAP
MS instrument name	ABI Sciex API 4000 QTrap
Ion Mode	POSITIVE
Units	nmol per mg tissue wt

Chromatography:

Chromatography ID:	CH003667
Instrument Name:	Sciex 4000 QTrap
Column Name:	none
Column Temperature:	none
Flow Gradient:	none
Flow Rate:	none
Solvent A:	none
Solvent B:	none
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS004602
Analysis ID:	AN004858
Instrument Name:	ABI Sciex API 4000 QTrap
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	see Mass_Spectrometry_Parameters.xlsx
Ion Mode:	POSITIVE
Acquisition Parameters File:	Mass_Spectrometry_Parameters.xlsx