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MB Sample ID: SA176343

Local Sample ID:KA-010
Subject ID:SU001980
Subject Type:Mammal
Subject Species:Homo sapiens
Taxonomy ID:9606

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Subject:

Subject ID:SU001980
Subject Type:Mammal
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
KA-010SA176343FL021880ControlTreatment

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
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