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MB Sample ID: SA176342
Local Sample ID: | KA-012 |
Subject ID: | SU001980 |
Subject Type: | Mammal |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001980 |
Subject Type: | Mammal |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
KA-012 | SA176342 | FL021880 | Control | Treatment |
Collection:
Collection ID: | CO001973 |
Collection Summary: | AsPC-1 PDAC cells were treated with DMSO vehicle control or 25 nM LGK974 (in triplicate per group). 1 x 10^6 cells were washed with ice-cold 150 mM ammonium acetate twice before adding 1 mL of ice-cold 80% methanol. After vigorous vortexing, samples were centrifuged at maximum speed, the aqueous layer was transferred to a glass vial and the metabolites were dried under vacuum. Metabolites were resuspended in 50 μL 70% acetonitrile (ACN) and 5 μL of this solution used for the mass spectrometer-based analysis. The analysis was performed on a Q Exactive (Thermo Fisher Scientific) in polarity-switching mode with positive voltage 4.0 kV and negative voltage 4.0 kV. The mass spectrometer was coupled to an UltiMate 3000RSLC (Thermo Fisher Scientific) UHPLC system. Mobile phase A was 5 mM ammonium acetate (NH4AcO), pH 9.9, B was acetonitrile and the separation achieved on a Luna 3 mm NH2 100 A column (150 × 2.0 mm, Phenomenex). The flow was 200 μL/min, and the gradient ran from 15% A to 95% A in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 min. Metabolites were detected and quantified as area under the curve based on retention time and accurate mass (≤ 3 p.p.m.) using the TraceFinder 3.1 (Thermo Fisher Scientific). Relative amounts of metabolites between various conditions, as well as percentage of [13C6] glucose labelling, were calculated and corrected for naturally occurring 13C abundance. |
Sample Type: | Cultured cells |
Treatment:
Treatment ID: | TR001992 |
Treatment Summary: | AsPC-1 PDAC cells were plated at 500,000 cells per well in 6 well plates in ultra-low attachment conditions. Cells were pre-treated with 25 nM LGK974 or DMSO vehicle control for 12 hours, maintained in normal media conditions. Media was exchanged with [13C6] glucose labeling media and cells were re-treated with 25 nM LGK974 or DMSO vehicle control for 24 hours. |
Sample Preparation:
Sampleprep ID: | SP001986 |
Sampleprep Summary: | Cells were washed with ice-cold 150 mM ammonium acetate twice before adding 1 mL of ice-cold 80% methanol. After vigorous vortexing, samples were centrifuged at maximum speed, the aqueous layer was transferred to a glass vial and the metabolites were dried under vacuum. Metabolites were resuspended in 50 μL 70% acetonitrile (ACN) and 5 μL of this solution used for the mass spectrometer-based analysis. The analysis was performed on a Q Exactive (Thermo Fisher Scientific) in polarity-switching mode with positive voltage 4.0 kV and negative voltage 4.0 kV. The mass spectrometer was coupled to an UltiMate 3000RSLC (Thermo Fisher Scientific) UHPLC system. Mobile phase A was 5 mM ammonium acetate (NH4AcO), pH 9.9, B was acetonitrile and the separation achieved on a Luna 3 mm NH2 100 A column (150 × 2.0 mm, Phenomenex). The flow was 200 μL/min, and the gradient ran from 15% A to 95% A in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 min. Metabolites were detected and quantified as area under the curve based on retention time and accurate mass (≤ 3 p.p.m.) using the TraceFinder 3.1 (Thermo Fisher Scientific). Relative amounts of metabolites between various conditions, as well as percentage of [13C6]glucose labeling, were calculated and corrected for naturally occurring 13C abundance. |
Combined analysis:
Analysis ID | AN003093 | AN003094 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Q Exactive | Q Exactive |
Column | Luna 3 mm NH2 100 A | Luna 3 mm NH2 100 A |
MS Type | API | API |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | area | area |
Chromatography:
Chromatography ID: | CH002284 |
Chromatography Summary: | Mobile phase A was 5 mM ammonium acetate (NH4AcO), pH 9.9, B was acetonitrile and the separation achieved on a Luna 3 mm NH2 100 A column (150 × 2.0 mm, Phenomenex). The flow was 200 μL/min, and the gradient ran from 15% A to 95% A in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 min. |
Instrument Name: | Q Exactive |
Column Name: | Luna 3 mm NH2 100 A |
Flow Rate: | 200 uL/min |
Solvent A: | 100% water; 5 mM ammonium acetate, pH 9.9 |
Solvent B: | 100% acetonitrile |
Chromatography Type: | HILIC |
MS:
MS ID: | MS002875 |
Analysis ID: | AN003093 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | API |
MS Comments: | Metabolites were detected and quantified as area under the curve based on retention time and accurate mass (≤ 3 p.p.m.) using the TraceFinder 3.1 (Thermo Fisher Scientific). Relative amounts of metabolites between various conditions, as well as percentage of [13C6] glucose labeling, were calculated and corrected for naturally occurring 13C abundance. |
Ion Mode: | POSITIVE |
Ion Spray Voltage: | 4000 |
MS ID: | MS002876 |
Analysis ID: | AN003094 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | API |
MS Comments: | Metabolites were detected and quantified as area under the curve based on retention time and accurate mass (≤ 3 p.p.m.) using the TraceFinder 3.1 (Thermo Fisher Scientific). Relative amounts of metabolites between various conditions, as well as percentage of [13C6] glucose labeling, were calculated and corrected for naturally occurring 13C abundance. |
Ion Mode: | NEGATIVE |
Ion Spray Voltage: | 4000 |