Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA254618

Local Sample ID:	T231
Subject ID:	SU002627
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002627
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
T231	SA254618	FL032625	NA	Genotype
T231	SA254618	FL032625	CB1158	Treatment

Collection:

Collection ID:	CO002620
Collection Summary:	Tumors were rapidly dissected after euthanizing animals. Tumors were weighed and rinsed in blood bank saline solution (150 mM NaCl) and blotted on filter paper (VWR, Radnor, PA, 28298–020). The process of dissection and tumor preparation took < 3min. Tumors were cut in half and put onto 20µm nylon mesh filters (Spectrum Labs, Waltham, MA, 148134) on top of 50 mL conical tubes, and centrifuged for 10min. at 4°C at 400xg. IF was then collected, snap-frozen in liquid nitrogen and stored at -80°C until further analysis.
Sample Type:	Interstitial Fluid

Treatment:

Treatment ID:	TR002639
Treatment Summary:	For genetically modified mice, LysM-Cre and Arg1fl/fl C57BL6J mice were bred to generate LysM-Cre +/+; Arg1fl/fl and litter mate control Arg1fl/fl mice. Orthotopic pancreatic cancer tumors were implanted in C57BL6J mice at 8-12 weeks of age. At endpoint, mice were euthanized by cervical dislocation, and tumors were harvested for TIF extraction. For Pharmacological inhibition, orthotopic tumors were implanted in C57BL6J mice at 8-12 weeks of age. 4 weeks after induction, once tumors were close end stage disease, CB-1158 (MedChem Express) dissolved in sterile water was administered by oral gavage at 100mg/kg. The acidity caused by the HCl in the drug solution was neutralized by adding equivalent amount of NaOH and an equivalent NaCl in sterile water solution was prepared as vehicle. 2hrs after treatment with CB-1158 or vehicle, mice were euthanized by cervical dislocation, and tumors were harvested for TIF extraction.

Treatment ID:

TR002639

Treatment Summary:

For genetically modified mice, LysM-Cre and Arg1fl/fl C57BL6J mice were bred to generate LysM-Cre +/+; Arg1fl/fl and litter mate control Arg1fl/fl mice. Orthotopic pancreatic cancer tumors were implanted in C57BL6J mice at 8-12 weeks of age. At endpoint, mice were euthanized by cervical dislocation, and tumors were harvested for TIF extraction. For Pharmacological inhibition, orthotopic tumors were implanted in C57BL6J mice at 8-12 weeks of age. 4 weeks after induction, once tumors were close end stage disease, CB-1158 (MedChem Express) dissolved in sterile water was administered by oral gavage at 100mg/kg. The acidity caused by the HCl in the drug solution was neutralized by adding equivalent amount of NaOH and an equivalent NaCl in sterile water solution was prepared as vehicle. 2hrs after treatment with CB-1158 or vehicle, mice were euthanized by cervical dislocation, and tumors were harvested for TIF extraction.

Sample Preparation:

Sampleprep ID:	SP002633
Sampleprep Summary:	polar metabolites were extracted from 5µL IF samples using 45µL 75:25:0.1 HPLC grade acetonitrile:methanol:formic acid extraction mix into which a mixture of isotopically labeled amino acids of known concentrations (Cambridge Isotope Laboratories, MSK-A2-1.2) was added. Samples were vortexed for 10 min, and centrifuged at maximum speed for 10 min. 30 μL of each extract was removed and dried under nitrogen gas and stored −80°C.

Sampleprep ID:

SP002633

Sampleprep Summary:

polar metabolites were extracted from 5µL IF samples using 45µL 75:25:0.1 HPLC grade acetonitrile:methanol:formic acid extraction mix into which a mixture of isotopically labeled amino acids of known concentrations (Cambridge Isotope Laboratories, MSK-A2-1.2) was added. Samples were vortexed for 10 min, and centrifuged at maximum speed for 10 min. 30 μL of each extract was removed and dried under nitrogen gas and stored −80°C.

Combined analysis:

Analysis ID	AN004161
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Dionex Ultimate 3000
Column	Waters XBridge Amide (100 x 4.6mm,3.5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	Peak area (m/z)

Chromatography:

Chromatography ID:	CH003080
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Column Temperature:	25
Flow Gradient:	0 min, 15% A; 2.5 min, 64% A; 12.4 min, 40% A; 12.5 min, 30% A; 12.5-14 min, 30% A; 14-21 min, 15% A
Flow Rate:	150 μL/min
Solvent A:	95% (vol/vol) water, 5% (vol/vol) acetonitrile, 10 mM ammonium hydroxide, 10 mM ammonium acetate, pH = 9.0
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS003908
Analysis ID:	AN004161
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	In positive/negative polarity switching mode, an m/z scan range from 60 to 900 was chosen and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 106 and the maximum injection time was 200 ms. The targeted ions were subsequently fragmented, using the higher energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500. Besides matching m/z, target metabolites are identified by matching either retention time with analytical standards and/or MS2 fragmentation pattern. Data acquisition and analysis were carried out by Xcalibur 4.1 software and Tracefinder 4.1 software, respectively (both from Thermo Fisher Scientific).
Ion Mode:	UNSPECIFIED