Global metabolic profiling of animal and human tissues via UPLC-MS

• Published in: Nature protocols Vol. 8; no. 1; pp. 17 - 32
• Main Authors: Want, Elizabeth J, Masson, Perrine, Michopoulos, Filippos, Wilson, Ian D, Theodoridis, Georgios, Plumb, Robert S, Shockcor, John, Loftus, Neil, Holmes, Elaine, Nicholson, Jeremy K
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-01-2013; Nature Publishing Group
• Subjects: 631/1647/2196/1380; 631/1647/320; 631/1647/527/296; Access to information; Analytical Chemistry; Analytical methods; Animal tissues; Animals; Biological Techniques; Biomarkers; Chromatography; Chromatography, Liquid - methods; Computational Biology/Bioinformatics; Data processing; High performance liquid chromatography; Humans; Life Sciences; Liquid chromatography; Liver - metabolism; Mass spectrometry; Mass Spectrometry - methods; Metabolism; Metabolites; Metabolome; Methods; Microarrays; Multivariate Analysis; Organic Chemistry; Physiological aspects; Physiology; protocol; Sample preparation; Scientific imaging; Tandem Mass Spectrometry - methods; United Kingdom; United Kingdom > UK
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2012.135

Obtaining comprehensive, untargeted metabolic profiles for complex solid samples, e.g., animal tissues, requires sample preparation and access to information-rich analytical methodologies such as mass spectrometry (MS). Here we describe a practical two-step process for tissue samples that is based on extraction into 'aqueous' and 'organic' phases for polar and nonpolar metabolites. Separation methods such as ultraperformance liquid chromatography (UPLC) in combination with MS are needed to obtain sufficient resolution to create diagnostic metabolic profiles and identify candidate biomarkers. We provide detailed protocols for sample preparation, chromatographic procedures, multivariate analysis and metabolite identification via tandem MS (MS/MS) techniques and high-resolution MS. By using these optimized approaches, analysis of a set of samples using a 96-well plate format would take ∼48 h: 1 h for system setup, 8–10 h for sample preparation, 34 h for UPLC-MS analysis and 2–3 h for preliminary/exploratory data processing, representing a robust method for untargeted metabolic screening of tissue samples.