Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA254583

Local Sample ID:	Plasma 3
Subject ID:	SU002626
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002626
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Plasma 3	SA254583	FL032620	Healthy_Mouse_Plasma	Sample type

Collection:

Collection ID:	CO002619
Collection Summary:	~100uL of blood were be obtained by submandibular sampling as described previously (Parasuraman et al., 2010) and centrifuged at 845 x g for 10 minutes at 4°C to separate plasma. Plasma was frozen in liquid nitrogen and stored at -80°C until further analysis animals. Mice were then immediately euthanized and tumor or pancreas from each animal was then harvested and immediately snap frozen using a BioSqueezer (BioSpec) cooled with liquid nitrogen and stored at -80°F until further analysis.
Sample Type:	Tumor, Pancreas & Plasma

Treatment:

Treatment ID:	TR002638
Treatment Summary:	Orthotopic tumors were implanted in C57BL6J mice at 8-12 weeks of age. 4 weeks after induction tumor-bearing mice and healthy littermate controls were treated with 15N2-glutamine (Cambridge Isotope Laboratory #NLM-1328-PK) dissolved in sterile phosphate buffered saline at 7.2mg/animal by tail vein injection as previously described (Lane et al., 2015). Briefly, animals were dosed three times at 15-minute intervals.

Treatment ID:

TR002638

Treatment Summary:

Orthotopic tumors were implanted in C57BL6J mice at 8-12 weeks of age. 4 weeks after induction tumor-bearing mice and healthy littermate controls were treated with 15N2-glutamine (Cambridge Isotope Laboratory #NLM-1328-PK) dissolved in sterile phosphate buffered saline at 7.2mg/animal by tail vein injection as previously described (Lane et al., 2015). Briefly, animals were dosed three times at 15-minute intervals.

Sample Preparation:

Sampleprep ID:	SP002632
Sampleprep Summary:	Cryogenically frozen tumor pieces were ground to a fine homogenous powder with a liquid nitrogen cooled mortar and pestle. ~30mg of tissue powder was weighed into sample tubes, and metabolites were extracted with 600µL HPLC grade methanol, 300µL HPLC grade water, and 400µL chloroform. Samples were vortexed for 10min at 4°C, centrifuged 21,000xg at 4°C for 10 min. 400µL of the aqueous top layer was removed into a new tube and dried under nitrogen. Dried tumor extracts were resuspended in 100µL HPLC grade water and LC-MS analysis was performed as described before(Sullivan et al., 2019b, 2019a). For plasma samples, we extracted polar metabolites from 5µL of sample using 45µL of a 75:25:0.1 HPLC grade acetonitrile:methanol:formic acid extraction mix. Samples in extraction mix were vortexed for 10 min at 4°C and centrifugated at 15,000x rpm for 10 min at 4°C to pellet insoluble material. 20µL of the soluble polar metabolite supernatant was moved to sample vials for analysis by LC-MS.

Sampleprep ID:

SP002632

Sampleprep Summary:

Cryogenically frozen tumor pieces were ground to a fine homogenous powder with a liquid nitrogen cooled mortar and pestle. ~30mg of tissue powder was weighed into sample tubes, and metabolites were extracted with 600µL HPLC grade methanol, 300µL HPLC grade water, and 400µL chloroform. Samples were vortexed for 10min at 4°C, centrifuged 21,000xg at 4°C for 10 min. 400µL of the aqueous top layer was removed into a new tube and dried under nitrogen. Dried tumor extracts were resuspended in 100µL HPLC grade water and LC-MS analysis was performed as described before(Sullivan et al., 2019b, 2019a). For plasma samples, we extracted polar metabolites from 5µL of sample using 45µL of a 75:25:0.1 HPLC grade acetonitrile:methanol:formic acid extraction mix. Samples in extraction mix were vortexed for 10 min at 4°C and centrifugated at 15,000x rpm for 10 min at 4°C to pellet insoluble material. 20µL of the soluble polar metabolite supernatant was moved to sample vials for analysis by LC-MS.

Combined analysis:

Analysis ID	AN004160
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Dionex Ultimate 3000
Column	SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE
Units	Fractional labelling

Chromatography:

Chromatography ID:	CH003079
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)
Column Temperature:	25
Flow Gradient:	linear gradient from 80% to 20% B; 20–20.5 min: linear gradient from 20% to 80% B; 20.5–28 min: hold at 80% B
Flow Rate:	0.150 mL/min
Solvent A:	20 mM ammonium carbonate, 0.1% ammonium hydroxide
Solvent B:	acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS003907
Analysis ID:	AN004160
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	XCalibur 965 2.2 software (Thermo 966 Fisher Scientific) was used identification and relative quantification for metabolites. Natural abundance correction was performed using the IsoCor (Millard et al., 2019).
Ion Mode:	POSITIVE