Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA358130

Local Sample ID:	control4
Subject ID:	SU003421
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

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Subject:

Subject ID:	SU003421
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
control4	SA358130	FL041791	Blood serum	Sample source
control4	SA358130	FL041791	WT	Genotype
control4	SA358130	FL041791	control	Treatment

Collection:

Collection ID:	CO003414
Collection Summary:	Blood was collected from control or overexpression of SLC7A14 in POMC neuron mice.Blood sample was collected into a BD Vacutainer tube not containing any anticoagulant, which was allowed to sit for ~30–60 min for clots to form following which serum was obtained by centrifugation (15 min at 4°C at 3500 rpm).
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR003430
Treatment Summary:	To overexpression of SLC7A14 in ARC POMC neurons, POMC Cre mice were bilaterally injected either with a Cre-dependent AAV vector containing SLC7A14 in the opposite orientation flanked by two inverted loxP sites (AAV9-Syn-DIO-SLC7A14-mCherry, 1.5 × 1012 Pfu/mL, HANBIO) at a volume of 200 nL into the ARC or an AAV vector containing only mCherry in the opposite orientation flanked by two inverted loxP sites (AAV9-Syn-DIO-mCherry, 1.5 × 1012 Pfu/mL, HANBIO) as a control. Serum was collected from control or overexpression of SLC7A14 in POMC neuron mice.

Treatment ID:

TR003430

Treatment Summary:

To overexpression of SLC7A14 in ARC POMC neurons, POMC Cre mice were bilaterally injected either with a Cre-dependent AAV vector containing SLC7A14 in the opposite orientation flanked by two inverted loxP sites (AAV9-Syn-DIO-SLC7A14-mCherry, 1.5 × 1012 Pfu/mL, HANBIO) at a volume of 200 nL into the ARC or an AAV vector containing only mCherry in the opposite orientation flanked by two inverted loxP sites (AAV9-Syn-DIO-mCherry, 1.5 × 1012 Pfu/mL, HANBIO) as a control. Serum was collected from control or overexpression of SLC7A14 in POMC neuron mice.

Sample Preparation:

Sampleprep ID:	SP003428
Sampleprep Summary:	The samples (100 μL) were placed in the EP tubes and resuspended with prechilled 80% methanol and 0.1% formic acid by well vortex. Then the sampleswere incubated on ice for 5 min and centrifuged at 15,000 g, 4°C for 20 min. Some of supernatant was diluted to final concentration containing 53% methanol by LC-MS grade water.The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15000 g, 4°C for 20 min. Finally, the supernatant was injected into the LC-MS/MS system analysis

Sampleprep ID:

SP003428

Sampleprep Summary:

The samples (100 μL) were placed in the EP tubes and resuspended with prechilled 80% methanol and 0.1% formic acid by well vortex. Then the sampleswere incubated on ice for 5 min and centrifuged at 15,000 g, 4°C for 20 min. Some of supernatant was diluted to final concentration containing 53% methanol by LC-MS grade water.The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15000 g, 4°C for 20 min. Finally, the supernatant was injected into the LC-MS/MS system analysis

Combined analysis:

Analysis ID	AN005407
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF-X Orbitrap
Ion Mode	NEGATIVE
Units	Peak area

Chromatography:

Chromatography ID:	CH004100
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um)
Column Temperature:	40°C
Flow Gradient:	2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min；100-2% B, 14.1 min；2% B, 17 min.
Flow Rate:	0.2 mL/min
Solvent A:	100% Water; 5mM Ammonium acetate
Solvent B:	100% Methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005134
Analysis ID:	AN005407
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.1 (CD3.1, ThermoFisher) to perform peak alignment, peak picking, and quantitation for each metabolite. The main parameterswere set as follows: retention time tolerance, 0.2 minutes; actual mass tolerance, 5ppm; signal intensity tolerance, 30%; signal/noise ratio, 3; and minimum intensity, et al. After that, peak intensities were normalized to the total spectral intensity.The normalized data was used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. And then peaks were matched with the mzCloud (https://www.mzcloud.org/)，mzVault and MassList database to obtain the accurate qualitative and relative quantitative results.Statistical analyses were performed using the statistical software R (R version R-3.4.3),Python (Python 2.7.6 version) and CentOS (CentOS release 6.6),When data were not normally distributed, normal transformations were attempted using of area normalization method.
Ion Mode:	NEGATIVE