Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA254676

Local Sample ID:	A35097_tumor_Control
Subject ID:	SU002629
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002629
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
A35097_tumor_Control	SA254676	FL032631	Tumor	Sample type
A35097_tumor_Control	SA254676	FL032631	Arg1fl/fl	Condition

Collection:

Collection ID:	CO002622
Collection Summary:	6~8-month-old female & male Lyz2-Cre +/+; Arg1fl/fl and litter mate control Arg1fl/fl mice with murine PDAC orthotopic tumors underwent dual jugular vein & carotid artery catheterization surgery. On day 5 of post recovery, mice received a 0.28 mg/g 10 min. bolus followed by a continuous 4 hr. infusion 0.005 mg/g/min infusion of 15N2-glutamine (Cambridge Isotope Laboratory #NLM-1328-PK). Plasma samples were taken at time points: 0' 15' 30' 60' 120' 180’ 240’. Tumors and tissues were harvested at 240’ and immediately snap frozen with liquid nitrogen stored at -80°C prior to analysis.
Sample Type:	Tumor, plasma

Treatment:

Treatment ID:	TR002641
Treatment Summary:	6~8-month-old female & male Lyz2-Cre +/+; Arg1fl/fl and litter mate control Arg1fl/fl mice with murine PDAC orthotopic tumors underwent dual jugular vein & carotid artery catheterization surgery. On day 5 of post recovery, mice received a 0.28 mg/g 10 min. bolus followed by a continuous 4 hr. infusion 0.005 mg/g/min infusion of 15N2-glutamine (Cambridge Isotope Laboratory #NLM-1328-PK). Plasma samples were taken at time points: 0' 15' 30' 60' 120' 180’ 240’. Tumors and tissues were harvested at 240’ and immediately snap frozen with liquid nitrogen stored at -80°C prior to analysis.

Treatment ID:

TR002641

Treatment Summary:

6~8-month-old female & male Lyz2-Cre +/+; Arg1fl/fl and litter mate control Arg1fl/fl mice with murine PDAC orthotopic tumors underwent dual jugular vein & carotid artery catheterization surgery. On day 5 of post recovery, mice received a 0.28 mg/g 10 min. bolus followed by a continuous 4 hr. infusion 0.005 mg/g/min infusion of 15N2-glutamine (Cambridge Isotope Laboratory #NLM-1328-PK). Plasma samples were taken at time points: 0' 15' 30' 60' 120' 180’ 240’. Tumors and tissues were harvested at 240’ and immediately snap frozen with liquid nitrogen stored at -80°C prior to analysis.

Sample Preparation:

Sampleprep ID:	SP002635
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:

SP002635

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	AN004163
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	Waters Atlantis BEH Z-HILIC (150 x 2.1 mm, 2.5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Orbitrap IQ-X Tribrid
Ion Mode	UNSPECIFIED
Units	Natural abundance corrected percentages

Chromatography:

Chromatography ID:	CH003082
Instrument Name:	Thermo Vanquish
Column Name:	Waters Atlantis BEH Z-HILIC (150 x 2.1 mm, 2.5um)
Column Temperature:	30°C
Flow Gradient:	0 minute: 85% B, 0.5 minute: 85% B, 18 minutes: 20% B, 20 minutes: 20% B, 20.5 minutes: 85% B and 28 minutes: 85% B
Flow Rate:	0.2 mL/minute
Solvent A:	20 mM ammonium bicarbonate at pH 9.6, adjusted by ammonium hydroxide addition
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS003910
Analysis ID:	AN004163
Instrument Name:	Thermo Orbitrap IQ-X Tribrid
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data acquisition was done using the Xcalibur software (Thermo Scientific) in full-scan mode with a range of 70-1000 m/z at 60K. Metabolite identification was done by matching the retention time and MS/MS fragmentation to the reference standards. Data analysis was performed using Tracefinder 5.1 software (Thermo Scientific).
Ion Mode:	UNSPECIFIED