Untargeted metabolomics from biological sources using ultraperformance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)

• Published in: Journal of visualized experiments 2013-05 (75), p.e50433
• Main Authors: Snyder, Nathaniel W, Khezam, Maya, Mesaros, Clementina A, Worth, Andrew, Blair, Ian A
• Format: Article
• Language: English
• Subjects: Biochemistry; Biomarkers - analysis; Blood Chemical Analysis - methods; Cell Extracts - analysis; Cell Line, Tumor; Chromatography, High Pressure Liquid - methods; Glioblastoma - chemistry; Humans; Metabolomics - methods; Tandem Mass Spectrometry - methods; Tissue Extracts - analysis
• ISSN: 1940-087X; 1940-087X
• DOI: 10.3791/50433

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.