Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA237288

Local Sample ID:	m334
Subject ID:	SU002458
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	wild type C57BL6/J
Age Or Age Range:	8 weeks
Weight Or Weight Range:	25-30g
Gender:	Male
Animal Animal Supplier:	Animal Resource Centre, Western Australai
Animal Housing:	standard
Animal Light Cycle:	12 h light dark cycle
Animal Feed:	normal chow
Animal Water:	autoclaved

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Subject:

Subject ID:	SU002458
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	wild type C57BL6/J
Age Or Age Range:	8 weeks
Weight Or Weight Range:	25-30g
Gender:	Male
Animal Animal Supplier:	Animal Resource Centre, Western Australai
Animal Housing:	standard
Animal Light Cycle:	12 h light dark cycle
Animal Feed:	normal chow
Animal Water:	autoclaved

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
m334	SA237288	FL029378	Unfasted/CSF1-FC	Treatment

Collection:

Collection ID:	CO002451
Collection Summary:	Liver samples were obtained from the same region of the left lateral lobe for each mouse.
Collection Protocol Filename:	Irvine_methods.docx
Sample Type:	Liver

Treatment:

Treatment ID:	TR002470
Treatment Summary:	Groups of male mice were treated with 4 x daily injections of Colony Stimulating Factor (1), followed by normal feeding or a 24 h fast from day 6 to day 7.

Sample Preparation:

Sampleprep ID:	SP002464
Sampleprep Summary:	Metabolites were extracted from 50 µl serum using 150 µl methanol and 50 µl chloroform, including 13C-sorbitol and 13C,15N-valine internal standards, and 30 µl of clarified sera was prepared for GC-MS analysis. Liver metabolites were extracted from 20-40 mg liver tissue (sampled from the same region of the left lateral lobe) in 600 µl methanol:MilliQ water (3:1), including 13C-sorbitol and 13C,15N-valine internal standards. The tissue was cryomilled at 4°C. 110 µl chloroform was added to 440 µg homogenate which was transferred to a new tube and the sample thermomixed for 20 minutes then centrifuged at 13,000 rpm for 15 minutes. 30 µl supernatant was dried in inserts for GC-MS analysis. Dried samples for targeted analysis were derivatised online using the Shimadzu AOC6000 autosampler robot. Derivatisation was achieved by the addition of 25 µL methoxyamine hydrochloride (30 mg/mL in pyridine, Merck) followed by shaking at 37°C for 2h. Samples were then derivatised with 25 µL of N,O-bis (trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA with 1% TMCS, Thermo Scientific) for 1h at 37°C. The sample was allowed to equilibrate at room temperature for 1 h before 1 µL was injected onto the GC column using a hot needle technique. Split (1:10) injections were performed for each sample.

Sampleprep ID:

SP002464

Sampleprep Summary:

Metabolites were extracted from 50 µl serum using 150 µl methanol and 50 µl chloroform, including 13C-sorbitol and 13C,15N-valine internal standards, and 30 µl of clarified sera was prepared for GC-MS analysis. Liver metabolites were extracted from 20-40 mg liver tissue (sampled from the same region of the left lateral lobe) in 600 µl methanol:MilliQ water (3:1), including 13C-sorbitol and 13C,15N-valine internal standards. The tissue was cryomilled at 4°C. 110 µl chloroform was added to 440 µg homogenate which was transferred to a new tube and the sample thermomixed for 20 minutes then centrifuged at 13,000 rpm for 15 minutes. 30 µl supernatant was dried in inserts for GC-MS analysis. Dried samples for targeted analysis were derivatised online using the Shimadzu AOC6000 autosampler robot. Derivatisation was achieved by the addition of 25 µL methoxyamine hydrochloride (30 mg/mL in pyridine, Merck) followed by shaking at 37°C for 2h. Samples were then derivatised with 25 µL of N,O-bis (trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA with 1% TMCS, Thermo Scientific) for 1h at 37°C. The sample was allowed to equilibrate at room temperature for 1 h before 1 µL was injected onto the GC column using a hot needle technique. Split (1:10) injections were performed for each sample.

Combined analysis:

Analysis ID	AN003864
Analysis type	MS
Chromatography type	None (Direct infusion)
Chromatography system	Shimadzu TQ8050NX
Column	Agilent DB5-MS (30m)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Shimadzu TQ8050NX
Ion Mode	UNSPECIFIED
Units	area

Chromatography:

Chromatography ID:	CH002862
Instrument Name:	Shimadzu TQ8050NX
Column Name:	Agilent DB5-MS (30m)
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS003605
Analysis ID:	AN003864
Instrument Name:	Shimadzu TQ8050NX
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The GC-MS system used comprised of an AOC6000 autosampler, a 2030 Shimadzu gas chromatograph and a TQ8050NX triple quadrupole mass spectrometer (Shimadzu, Japan). The mass spectrometer was tuned according to the manufacturer’s recommendations using tris-(perfluorobutyl)-amine (CF43). GC-MS was performed on a 30m Agilent DB-5 column with 0.25mm internal diameter column and 1µm film thickness. The injection temperature (inlet) was set at 280°C, the MS transfer line at 280°C and the ion source adjusted to 200°C. Helium was used as the carrier gas at a flow rate of 1 mL/min and argon gas was used in the collision cell to generate the MRM product ion. The analysis of TMS samples was performed under the following oven temperature program; 100°C start temperature, hold for 4 minutes, followed by a 10°C min-1 oven temperature ramp to 320°C with a following final hold for 11 minutes. Approximately 520 targets were collected using the Shimadzu Smart Metabolite Database, where each target comprised a quantifier MRM along with a qualifier MRM, which covers approximately 350 endogenous metabolites and multiple stable isotopically labelled internal standards. Resultant data was processed using Shimadzu LabSolutions Insight software, where peak integrations were visually validated and manually corrected where required.
Ion Mode:	UNSPECIFIED