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Nat Protoc. 2013 Jan;8(1):17-32. doi: 10.1038/nprot.2012.135. Epub 2012 Dec 6.

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

Author information

1
Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College, London, UK.

Abstract

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.

PMID:
23222455
DOI:
10.1038/nprot.2012.135
[Indexed for MEDLINE]

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