Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA308653

Local Sample ID:	tumor-veh-3-01
Subject ID:	SU002964
Subject Type:	Mammal
Subject Species:	Mus musculus
Genotype Strain:	NSG

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Subject:

Subject ID:	SU002964
Subject Type:	Mammal
Subject Species:	Mus musculus
Genotype Strain:	NSG

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
tumor-veh-3-01	SA308653	FL037167	Vehicle	Treatment
tumor-veh-3-01	SA308653	FL037167	1	Technical Replicate (Region of Tumor)

Collection:

Collection ID:	CO002957
Collection Summary:	Tumors were collected after 10 days of treatment and prepared for histology, protein extraction, and metabolite extraction.
Sample Type:	Prostate

Treatment:

Treatment ID:	TR002973
Treatment Summary:	7 million 16D cells were implanted subcutaneously with 100 μl Matrigel (Corning) into NSG mice to form primary tumors. Primary tumors were harvested, minced, and re-implanted (20 - 80 mg of minced tumor tissue with 100 μl Matrigel per mouse) into NSG mice. 16D tumor-bearing mice were treated by oral gavage with 10 mg/kg/day of Enzalutamide in the vehicle (1% carboxymethyl cellulose, 0.5% Tween 80, and 5% dimethylsulfoxide) or the vehicle only, with a two-days-on/one-day-off schedule.

Treatment ID:

TR002973

Treatment Summary:

7 million 16D cells were implanted subcutaneously with 100 μl Matrigel (Corning) into NSG mice to form primary tumors. Primary tumors were harvested, minced, and re-implanted (20 - 80 mg of minced tumor tissue with 100 μl Matrigel per mouse) into NSG mice. 16D tumor-bearing mice were treated by oral gavage with 10 mg/kg/day of Enzalutamide in the vehicle (1% carboxymethyl cellulose, 0.5% Tween 80, and 5% dimethylsulfoxide) or the vehicle only, with a two-days-on/one-day-off schedule.

Sample Preparation:

Sampleprep ID:	SP002970
Sampleprep Summary:	After tumor dissection, a maximum of 30mg of tissue was weighed, snap frozen, and stored at -80C until metabolite extraction. To extract metabolites, weighed tumor tissue was added to a bead tube (Thermo Fisher Scientific) containing 1ml 80% methanol plus 10mM potassium trifluoromethanesulfonate (TMSO) internal standard on ice. Samples were homogenized for 1 minute at max speed on a bead homogenizer (Thermo Fisher Scientific). Bead tubes were spun at 17000g at 4C for 10 minutes. The supernatant was transferred to an Eppendorf tube and spun at 17000g at 4C for 10 minutes. A volume of extraction equivalent to 3mg of tumor tissue was transferred to an ABC vial (Thermo Fisher Scientific). All volumes were normalized to 500 µl with 80% methanol containing TMSO internal standard. 80% MeOH was evaporated from the ABC vials using the EZ-2Elite evaporator (Genevac) and samples were stored at -80°C until analysis. Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile (ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 minutes at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials. 10 µL of these metabolite solutions were injected per analysis.

Sampleprep ID:

SP002970

Sampleprep Summary:

After tumor dissection, a maximum of 30mg of tissue was weighed, snap frozen, and stored at -80C until metabolite extraction. To extract metabolites, weighed tumor tissue was added to a bead tube (Thermo Fisher Scientific) containing 1ml 80% methanol plus 10mM potassium trifluoromethanesulfonate (TMSO) internal standard on ice. Samples were homogenized for 1 minute at max speed on a bead homogenizer (Thermo Fisher Scientific). Bead tubes were spun at 17000g at 4C for 10 minutes. The supernatant was transferred to an Eppendorf tube and spun at 17000g at 4C for 10 minutes. A volume of extraction equivalent to 3mg of tumor tissue was transferred to an ABC vial (Thermo Fisher Scientific). All volumes were normalized to 500 µl with 80% methanol containing TMSO internal standard. 80% MeOH was evaporated from the ABC vials using the EZ-2Elite evaporator (Genevac) and samples were stored at -80°C until analysis. Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile (ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 minutes at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials. 10 µL of these metabolite solutions were injected per analysis.

Combined analysis:

Analysis ID	AN004672	AN004673
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Peak Area	Peak Area

Chromatography:

Chromatography ID:	CH003516
Chromatography Summary:	Samples were run on a Vanquish (Thermo Fisher Scientific) UHPLC system with mobile phase A (20mM ammonium carbonate, pH 9.7) and mobile phase B (100% ACN) at a flow rate of 150 µL/min on a SeQuant ZIC-pHILIC Polymeric column (2.1 × 150 mm 5 μm, EMD Millipore) at 35°C. Separation was achieved with a linear gradient from 20% A to 80% A in 20 minutes followed by a linear gradient from 80% A to 20% A from 20 minutes to 20.5 minutes. 20% A was then held from 20.5 minutes to 28 minutes.
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
Column Temperature:	35°C
Flow Gradient:	Linear gradient was as follows: 20%A to 80%A (0-20 min), 80%A to 20%A (20-20.5 min), hold 20%A (20.5-28.0 min).
Flow Rate:	150 µL/min
Solvent A:	20 mM Ammonium carbonate, pH 9.7
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS004419
Analysis ID:	AN004672
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC was coupled to a Q-Exactive (Thermo Fisher Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range=(70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard12. These “.mzXML” files were imported into the MZmine 2 software package13. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module14 and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor15 to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well.
Ion Mode:	POSITIVE

MS ID:	MS004420
Analysis ID:	AN004673
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC was coupled to a Q-Exactive (Thermo Fisher Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range=(70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard12. These “.mzXML” files were imported into the MZmine 2 software package13. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module14 and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor15 to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well.
Ion Mode:	NEGATIVE