Samples were prepared and analyzed in the Roswell Park Comprehensive Cancer Center Bioanalytics, Metabolomics and Pharmacokinetics Shared Resource, using the MxP Quant 500 kit (Biocrates Life Sciences AG) in accordance with the user manual. Tumor samples were homogenized in a ratio of 1 mg of tissue to 3 μL of solvent (85% ethanol and 15% 0.01 mol/L phosphate buffer) using optimized setting on the Omni-Bead Ruptor 24 (Omni International). The homogenate was centrifuged to obtain a supernatant which was added to the plate (10 μL). Sample extract elution was performed with 5-mmol/L ammonium acetate in methanol and diluted with either water for the high pressure liquid chromatography (HPLC)-MS/MS analysis (1:1) or kit running solvent (Biocrates Life Sciences AG) for flow injection analysis–MS/MS (50:1), using a Sciex 5500 mass spectrometer. Data was processed using MetIDQ software (Biocrates Life Sciences AG), Limma (33 (link)) for differential metabolites and Metaboanalyst (34 (link)) for metabolite set enrichment analysis.
Protocol detail
Metabolomic Profiling of Tumor Samples
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Ammonium acetate
Buffer
Cancer
Ethanol
Flow injection analysis
High pressure liquid chromatography
Methanol
Ms ms
Pharmacokinetics
Phosphate
Solvent
Tissue
Tumor
Corresponding Organization : Roswell Park Comprehensive Cancer Center
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Variable analysis
independent variables
- None explicitly mentioned
- Metabolite levels in tumor samples
- Tumor sample homogenization ratio (1 mg tissue : 3 μL solvent)
- Solvent composition (85% ethanol, 15% 0.01 mol/L phosphate buffer)
- Homogenization settings on Omni-Bead Ruptor 24
- Sample extract elution (5-mmol/L ammonium acetate in methanol)
- Sample dilution for HPLC-MS/MS (1:1) and flow injection analysis–MS/MS (50:1)
- Mass spectrometer (Sciex 5500)
- Data processing software (MetIDQ, Limma, Metaboanalyst)
- No positive or negative controls were explicitly mentioned in the input.
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