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MB Sample ID: SA194287
Local Sample ID: | KL_4 |
Subject ID: | SU002148 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57Bl/6 GEMMs |
Age Or Age Range: | Mutants: K: 90 days, KK: 60 days, KL and KKL: 40 days post Ad5-CMV-Cre |
Gender: | Male and female |
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Subject:
Subject ID: | SU002148 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57Bl/6 GEMMs |
Age Or Age Range: | Mutants: K: 90 days, KK: 60 days, KL and KKL: 40 days post Ad5-CMV-Cre |
Gender: | Male and female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
KL_4 | SA194287 | FL023820 | Kras/Lkb1 | treatment |
Collection:
Collection ID: | CO002141 |
Collection Summary: | At defined timepoints (K: 90 days, KK: 60 days, KL and KKL: 40 days post Ad5-CMV-Cre), mice were sacrificed by cardiac puncture and lungs and plasma harvested for downstream analysis. |
Sample Type: | Lung |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR002160 |
Treatment Summary: | Mice were genotyped using primers outlined in the Key Resources Table. Seven-to-eight-week old conditional mice were intranasally (i.n) infected with 20 ul of 1x1010 PFU/ml Ad5-CMV-Cre virus (University of Iowa Gene Transfer Core Facility) according to standard procedures62. For anti-PD1 survival study, 20 days following Ad5-CMV-Cre, KL and KKL mice were randomly assigned to anti-PD1 or isotype control antibody (200 g RMP1-14 or Rat IgG2a; BioXCell) treatment arms (n = 6/arm). Each cycle (every 20 days) consisted of intra-peritoneal injection (200 l into left and right flank) three times over 6 days (day 0, 3, 6). Mice were collected at ethical endpoint for Kaplan Meier survival analysis. For combination anti-PD1/CB-839 study, 20 days following Ad5-CMV-Cre, KL mice were randomly assigned to vehicle/isotype, vehicle/anti-PD1, CB-839/isotype or CB-839/anti-PD1 treatment arms (study 1: n = 6/arm; study 2: n = 4/arm). On day 20, mice received one cycle of anti-PD1 or isotype control (as above) and 200 mg/kg CB-839 (2 % w/v CB-839; BLDpharm, in vehicle) or vehicle (20 % Solutol HS15 / 20 % SB-β-CD (Captisol) / 50 mM phosphate buffer, pH 7.4) twice daily by oral gavage until collection on day 29, when mice were collected 12 hours following the final dose. At harvest, mice were sacrificed by cardiac bleed and the left lung lobe was weighed and processed for flow cytometry with spleens, while right lung lobes were inflated with 4 % paraformaldehyde and processed for histology. |
Treatment Compound: | 200 ug RMP1-14 or Rat IgG2a; Combination treatments: vehicle/isotype, vehicle/anti-PD1, CB-839/isotype or CB-839/anti-PD1 treatment arms |
Treatment Route: | by oral gavage |
Treatment Dose: | multiple dose 200 ug. Combination treatments: 200 mg/kg CB-839 (2 % w/v CB-839; BLDpharm, in vehicle) or vehicle (20 % Solutol HS15 / 20 % SB-β-CD (Captisol) / 50 mM phosphate buffer, pH 7.4) twice daily by oral gavage until collection on day 29, |
Sample Preparation:
Sampleprep ID: | SP002154 |
Sampleprep Summary: | Tumor tissue was flash frozen and crushed using a mortar and pestle in liquid nitrogen. To the Eppendorf tubes 150 µL of cold 80 % acetonitrile was added and vortexed quickly. The suspension was transferred to a fresh Eppendorf tube. Another 150 µL portion of extraction solvent was added to the tubes, vortexed and the suspension combined with the first portion. To the combined extraction mixture 75 µL of CHCl3 was added and samples were mixed for 1 h in a cool room to ensure complete extraction. The samples were centrifuged at 4 °C at 14.8 g for 10 min, supernatant transferred to new tubes and evaporated at 20 °C under a stream of nitrogen. BSA assay was performed on the protein pellet by dissolving all protein in equal volume of detergent solution (4 % v/v SDS in water). Dried samples were resolubilized in appropriate volume of CHCl3 : MeOH : H2O (1 : 3 :1, v/v) mixture based on measured protein content, shaken for 15 min at room temperature, centrifuged at 4 °C at 14.8 g for 10 min. 100 µL of the supernatant was transferred to LCMS vials. 10 µL injected for metabolomics analysis. |
Processing Storage Conditions: | -80℃ |
Extract Storage: | -80℃ |
Combined analysis:
Analysis ID | AN003365 | AN003366 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
Column | SeQuant ZIC-pHILIC (150 x 4.2mm,5um) | SeQuant ZIC-pHILIC (150 x 4.2mm,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 |
Chromatography:
Chromatography ID: | CH002489 |
Chromatography Summary: | LCMS data was acquired on Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) coupled with high-performance liquid chromatography (HPLC) system Dionex Ultimate® 3000 RS (Thermo Fisher). Chromatographic separation was performed on a ZIC-pHILIC column (5 µm, polymeric, 150 × 4.6 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate and (B) acetonitrile. The gradient program started at 80% B and was reduced to 50% B over 15 min, then reduced from 50% B to 5% B over 3 min, followed by wash with 5% B for another 3 min, and finally 8 min re-equilibration with 80% B. The flow rate was 0.3 mL/min and column compartment temperature was 40ºC. The total run time was 32 min with an injection sample volume of 10 µL. |
Methods Filename: | Metabolomics_pHILIC_Parkville_v1.meth |
Instrument Name: | Thermo Dionex Ultimate 3000 RS |
Column Name: | SeQuant ZIC-pHILIC (150 x 4.2mm,5um) |
Column Pressure: | 60 bar at starting conditions |
Column Temperature: | 25 C |
Flow Gradient: | 0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B |
Flow Rate: | 0.3 ml/min |
Injection Temperature: | 4 C |
Internal Standard: | CAPS, CHAPS, PIPES |
Sample Injection: | 10 uL |
Solvent A: | 100% water; 20 mM ammonium formate |
Solvent B: | 100% acetonitrile |
Analytical Time: | 32 min |
Capillary Voltage: | 3.5 kV |
Oven Temperature: | 25 C |
Washing Buffer: | syringe wash 50% IPA |
Weak Wash Solvent Name: | 10% MeOH |
Strong Wash Solvent Name: | 10% MeOH |
Sample Loop Size: | 25 uL |
Sample Syringe Size: | 25 uL |
Chromatography Type: | HILIC |
MS:
MS ID: | MS003132 |
Analysis ID: | AN003365 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. |
Ion Mode: | POSITIVE |
Capillary Temperature: | 300 C |
Capillary Voltage: | 4 kV |
Collision Energy: | NA |
Collision Gas: | NA |
Ion Source Temperature: | 120 C |
Mass Accuracy: | 1-2 ppm |
Automatic Gain Control: | 1e6 |
Dataformat: | profile |
Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
Analysis Protocol File: | PQMS3-MBPF-WIN-0501_analysis.pdf |
MS ID: | MS003133 |
Analysis ID: | AN003366 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. |
Ion Mode: | NEGATIVE |
Capillary Temperature: | 300 C |
Capillary Voltage: | 3.5 kV |
Collision Energy: | NA |
Collision Gas: | NA |
Ion Source Temperature: | 120 C |
Mass Accuracy: | 1-2 ppm |
Automatic Gain Control: | 1e6 |
Dataformat: | profile |
Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
Analysis Protocol File: | PQMS3-MBPF-WIN-0501_analysis.pdf |