Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA085810

Local Sample ID:	4801 03
Subject ID:	SU001280
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

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Subject:

Subject ID:	SU001280
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
4801 03	SA085810	FL012832	22C	Temperature
4801 03	SA085810	FL012832	KO	Genotype

Collection:

Collection ID:	CO001274
Collection Summary:	Mice were anaesthetised with Isoflurane (under controlled flow-rate), and placed in a hot pad, while the blood was drawn from the tail into a tube. The blood was left at 22C for 30 - 60 minutes, and then centrifuged at 14,000 RPM for 3 minutes. The serum fraction was collected and frost at -20C.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR001295
Treatment Summary:	Wild-type and Ucp1CRE/12-LOX KO mice were exposed to a short-term cold temperature (1 hour at 5C) or kept at room temperature. After this period, the serum was collected as described. Ucp1CRE/12-LOX KO mice were created through CRISPR-Cas9 technology, as described in Leiria et al., 2019, Cell Metabolism.

Sample Preparation:

Sampleprep ID:	SP001288
Sampleprep Summary:	Aliquots of 100 µL serum or 1mg protein from homogenized tissue (measured by BCA) were taken, depending on the experiment. A mixture of deuterium-labeled internal standards was added to each aliquot, followed by 3x volume of sample of cold methanol (MeOH). Samples were vortexed for 5 minutes and stored at 31 −20 °C overnight. Cold samples were centrifuged at 14,000g for 10 minutes, and the supernatant was then transferred to a new tube and 3 mL of acidified H 2 O (pH 3.5) was added to each sample prior to C18 SPE. The methyl formate fractions were collected, dried under nitrogen, and reconstituted in 50 µL MeOH:H 2 O (1:1, by vol). Samples were transferred to 0.5 mL tubes and centrifuged at 20,000g at 4 °C for 10 minutes. 35ul of supernatant was transferred to LC–MS/MS vials for analysis using the BERG LCMS/MS mediator lipidomics platform.

Sampleprep ID:

SP001288

Sampleprep Summary:

Aliquots of 100 µL serum or 1mg protein from homogenized tissue (measured by BCA) were taken, depending on the experiment. A mixture of deuterium-labeled internal standards was added to each aliquot, followed by 3x volume of sample of cold methanol (MeOH). Samples were vortexed for 5 minutes and stored at 31 −20 °C overnight. Cold samples were centrifuged at 14,000g for 10 minutes, and the supernatant was then transferred to a new tube and 3 mL of acidified H 2 O (pH 3.5) was added to each sample prior to C18 SPE. The methyl formate fractions were collected, dried under nitrogen, and reconstituted in 50 µL MeOH:H 2 O (1:1, by vol). Samples were transferred to 0.5 mL tubes and centrifuged at 20,000g at 4 °C for 10 minutes. 35ul of supernatant was transferred to LC–MS/MS vials for analysis using the BERG LCMS/MS mediator lipidomics platform.

Combined analysis:

Analysis ID	AN002024
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Ekspert MicroLC 200 system
Column	Phenomenex Synergi Fusion-RP capillary C18 (150 × 0.5 mm,4um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	ABI Sciex 5600+ TripleTOF
Ion Mode	NEGATIVE
Units	Peak Area

Chromatography:

Chromatography ID:	CH001465
Instrument Name:	Ekspert MicroLC 200 system
Column Name:	Phenomenex Synergi Fusion-RP capillary C18 (150 × 0.5 mm,4um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001877
Analysis ID:	AN002024
Instrument Name:	ABI Sciex 5600+ TripleTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	MS spectra were acquired in high-resolution mode (>30,000) using a 100-ms accumulation time per spectrum. Fullscan MS/MS was acquired in high sensitivity mode, with an accumulation time optimized per cycle. Collision energy was set using rolling collision energy with a spread of 15V. The identity of a component was confirmed using PeakView® software (SCIEX), and quantification was performed using MultiQuant™ software (SCIEX).
Ion Mode:	NEGATIVE