Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA272176

Local Sample ID:	PL11
Subject ID:	SU002811
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	B6.129-Arntltm1Bra/J
Age Or Age Range:	40 weeks
Gender:	Male
Animal Feed:	Ad libitum
Animal Water:	Ad libitum

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Subject:

Subject ID:	SU002811
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	B6.129-Arntltm1Bra/J
Age Or Age Range:	40 weeks
Gender:	Male
Animal Feed:	Ad libitum
Animal Water:	Ad libitum

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
PL11	SA272176	FL035255	Bmal1-KO	Genotype
PL11	SA272176	FL035255	AAV	Treatment

Collection:

Collection ID:	CO002804
Collection Summary:	Mice were anesthetized using isofluorane. Blood was collected by intracardiac puncture. EDTA was used as anticoagulant. Blood was centrifuged at 2,000xg for 15 mins and plasma was collected and subsequently frozen in liquid nitrogen. Samples are stored at -80°C.
Sample Type:	Blood (plasma)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002820
Treatment Summary:	Bmal1-KO+AAV mice were systemically infected in the subxiphoid region with 20μl containing 2 × 10^11 genome copies of AAV9 on postnatal day 5.

Sample Preparation:

Sampleprep ID:	SP002817
Sampleprep Summary:	All provided samples were extracted following our cellular extraction procedure with pre-normalization to the sample protein content. Each extract was spiked with 1 ul of standard mixture consisting of deuterium labeled carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, myristoylcarnitine and palmitoylcarnitine to targeted identification of carnitines in the sample. Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein precipitation was allowed by incubating the samples at 4°C for 20 min. Samples were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from each sample into clean tube and dried under a gentle stream of nitrogen at 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic acid. Resuspension was allowed at 4°C for 10 -15 min then samples were centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler.

Sampleprep ID:

SP002817

Sampleprep Summary:

All provided samples were extracted following our cellular extraction procedure with pre-normalization to the sample protein content. Each extract was spiked with 1 ul of standard mixture consisting of deuterium labeled carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, myristoylcarnitine and palmitoylcarnitine to targeted identification of carnitines in the sample. Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein precipitation was allowed by incubating the samples at 4°C for 20 min. Samples were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from each sample into clean tube and dried under a gentle stream of nitrogen at 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic acid. Resuspension was allowed at 4°C for 10 -15 min then samples were centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler.

Combined analysis:

Analysis ID	AN004387	AN004388
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Dionex	Thermo Dionex
Column	ACE Excel 2 C18-PFP (100 x 2.1mm,2um)	ACE Excel 2 C18-PFP (100 x 2.1mm,2um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Peak height	Peak height

Chromatography:

Chromatography ID:	CH003291
Chromatography Summary:	Global metabolomics profiling was performed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. Each extract was spiked with 1 ul of standard mixture consisting of deuterium labeled carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, myristoylcarnitine and palmitoylcarnitine to targeted identification of carnitines in the sample. All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 2 Excel-C18 pfp, 100x2.1mm, 2um column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is a polar embedded stationary phase that provides comprehensive coverage, but does have some limitation is the coverage of very polar species. The flow rate was 350 µL/min with a column temperature of 25°C. 4 µL was injected for negative ions and 2 µL for positive ions.
Instrument Name:	Thermo Dionex
Column Name:	ACE Excel 2 C18-PFP (100 x 2.1mm,2um)
Column Temperature:	25°C
Flow Gradient:	-
Flow Rate:	350 µL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile
Chromatography Type:	Reversed phase
Solvent C:	-

MS:

MS ID:	MS004136
Analysis ID:	AN004387
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 µm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is a polar embedded stationary phase that provides comprehensive coverage, but does have some limitation is the coverage of very polar species. The flow rate was 350 µL/min with a column temperature of 25°C. 4 µL was injected for negative ions and 2 µL for positive ions.
Ion Mode:	POSITIVE

MS ID:	MS004137
Analysis ID:	AN004388
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 µm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is a polar embedded stationary phase that provides comprehensive coverage, but does have some limitation is the coverage of very polar species. The flow rate was 350 µL/min with a column temperature of 25°C. 4 µL was injected for negative ions and 2 µL for positive ions.
Ion Mode:	NEGATIVE