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MB Sample ID: SA193912
Local Sample ID: | 4T1-25_R16+ |
Subject ID: | SU002140 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002140 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
4T1-25_R16+ | SA193912 | FL023742 | Lung | Tissue |
4T1-25_R16+ | SA193912 | FL023742 | Tumor | Treatment |
Collection:
Collection ID: | CO002133 |
Collection Summary: | Lung and skeletal muscle (TA and bulk muscles from the upper arm) were collected upon necropsy and imaged for mCherry+ 4T1-SkM lesions (Zeiss AxioZoom). Once a mCherry+ lesion was identified, the region was excised, leaving as little tissue margins as possible. The sample was weighed and then flash frozen using LN2. Healthy lung and skeletal muscle samples were collected through the same process, without the mCherry imaging step. 4T1-parental and 4T1-SkM cells were plated in 6-well plates at an initial density of 50,000 cells/well and collected 48h later (final density ~275,000 cells/well). Wells were washed twice with 1x PBS and cells were scraped into 1.5 mL microcentrifuge tubes and spun at 16,000 x g for 3 minutes. Samples were then flash frozen and stored at -80°C until ready to process. |
Sample Type: | Lung |
Treatment:
Treatment ID: | TR002152 |
Treatment Summary: | Lung and skeletal muscle (TA and bulk muscles from the upper arm) were collected upon necropsy and imaged for mCherry+ 4T1-SkM lesions (Zeiss AxioZoom). Once a mCherry+ lesion was identified, the region was excised, leaving as little tissue margins as possible. The sample was weighed and then flash frozen using LN2. Healthy lung and skeletal muscle samples were collected through the same process, without the mCherry imaging step. 4T1-parental and 4T1-SkM cells were plated in 6-well plates at an initial density of 50,000 cells/well and collected 48h later (final density ~275,000 cells/well). Wells were washed twice with 1x PBS and cells were scraped into 1.5 mL microcentrifuge tubes and spun at 16,000 x g for 3 minutes. Samples were then flash frozen and stored at -80°C until ready to process. |
Sample Preparation:
Sampleprep ID: | SP002146 |
Sampleprep Summary: | Prior to LC-MS analysis, tissue samples were weighed and resuspended in pre-chilled (-20°C) methanol:acetonitrile:water (5:3:2, v:v) to a final concentration of 30 mg/ml, cell samples were placed on ice and re-suspended with methanol:acetonitrile:water (5:3:2, v:v) at a concentration of 2x106 cells/mL, and media samples were extracted with the same solution at a dilution of 1:25 (v/v). Suspensions were vortexed continuously for 30 min at 4°C. Insoluble material was removed by centrifugation at 18,000 g for 10 min at 4°C and supernatants were isolated for metabolomics analysis by UHPLC-MS. Analyses were performed as previously published85,86. Injection volumes for tissue, cell, and media extracts were 2 L, 10 L, and 10L, respectively. The analytical platform employs a Vanquish UHPLC system (ThermoFisher) coupled online to a Q Exactive mass spectrometer (ThermoFisher). Samples were resolved over a Kinetex C18 column, 2.1 x 150 mm, 1.7 m particle size (Phenomenex, Torrance, CA) equipped with a guard column (SecurityGuardTM Ultracartridge – UHPLC C18 for 2.1 mm ID Columns – AJO-8782 – Phenomenex, Torrance, CA) using an aqueous phase (A) of water and 0.1% formic acid and a mobile phase (B) of acetonitrile and 0.1% formic acid for positive ion polarity mode, and an aqueous phase (A) of water:acetonitrile (95:5) with 1 mM ammonium acetate, and a mobile phase (B) of acetonitrile:water (95:5) with 1 mM ammonium acetate for negative ion polarity mode. Samples were eluted from the column using either an isocratic elution of 5% B flowed at 250 L/min and 25°C or a gradient from 5% to 95% B over 1 min, followed by an isocratic hold at 95% B for 2 min, flowed at 400 L/min and 45°C. The Q Exactive mass spectrometer (ThermoFisher) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 scans) from 60 to 900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. Calibration was performed prior to analysis using the PierceTM Positive and Negative Ion Calibration Solutions (ThermoFisher). Acquired data was then converted from raw to mzXML file format using Mass Matrix (Cleveland, OH). Samples were analyzed in randomized order with a technical mixture injected periodically through analysis to qualify instrument performance. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ)92. |
Combined analysis:
Analysis ID | AN003350 | AN003351 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | QExactive | QExactive |
Column | Kinetix C18 (100 x 2.1mm) | Kinetix C18 (100 x 2.1mm) |
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 | AU | AU |
Chromatography:
Chromatography ID: | CH002481 |
Instrument Name: | QExactive |
Column Name: | Kinetix C18 (100 x 2.1mm) |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS003119 |
Analysis ID: | AN003350 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The Q Exactive mass spectrometer (ThermoFisher) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 scans) from 60 to 900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. Calibration was performed prior to analysis using the PierceTM Positive and Negative Ion Calibration Solutions (ThermoFisher). Acquired data was then converted from raw to mzXML file format using Mass Matrix (Cleveland, OH). Samples were analyzed in randomized order with a technical mixture injected periodically through analysis to qualify instrument performance. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ) |
Ion Mode: | POSITIVE |
MS ID: | MS003120 |
Analysis ID: | AN003351 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The Q Exactive mass spectrometer (ThermoFisher) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 scans) from 60 to 900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. Calibration was performed prior to analysis using the PierceTM Positive and Negative Ion Calibration Solutions (ThermoFisher). Acquired data was then converted from raw to mzXML file format using Mass Matrix (Cleveland, OH). Samples were analyzed in randomized order with a technical mixture injected periodically through analysis to qualify instrument performance. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ) |
Ion Mode: | NEGATIVE |