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. 2012 Apr:Chapter 30:Unit 30.2.1-24.
doi: 10.1002/0471142727.mb3002s98.

Targeted metabolomics

Lee D Roberts  1 , Amanda L Souza, Robert E Gerszten, Clary B Clish
Affiliations

Targeted metabolomics

Lee D Roberts et al. Curr Protoc Mol Biol. 2012 Apr.

Abstract

The metabolome is the terminal downstream product of the genome and consists of the total complement of all the low-molecular-weight molecules (metabolites) in a cell, tissue, or organism. Metabolomics aims to measure a wide breadth of small molecules in the context of physiological stimuli or disease states. Metabolomics methodologies fall into two distinct groups: untargeted metabolomics, an intended comprehensive analysis of all the measurable analytes in a sample including chemical unknowns, and targeted metabolomics, the measurement of defined groups of chemically characterized and biochemically annotated metabolites. The methodologies considered in this unit focus on the processes of conducting targeted metabolomics experiments, and the advantages of this general approach are highlighted herein. This unit outlines procedures for extracting nitrogenous metabolites (including amino acids), lipids, and intermediary metabolites (including TCA cycle oxoacids) from blood plasma. Specifically, protocols are described for analyzing these metabolites using targeted metabolomics experiments based on liquid chromatography-mass spectrometry.

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Figures

Figure 30.2.1
Figure 30.2.1
The relationship between the genome, proteome and metabolome (Wang and Gerszten, Nature, 2008).
Figure 30.2.2
Figure 30.2.2
(A) Workflow for a Liquid Chromatography Triple Quadrupole Mass Spectrometry Multiple Reaction Monitoring targeted metabolomics experiment. (B) The resolution of chromatography allows the separation of metabolites even in the case of a shared MRM transition. (C) Specific precursor/product ion transitions in MRM experiments identify individual metabolites.
Figure 30.2.3
Figure 30.2.3
An example of an experiment to assess biological and technical variance (left of diagram), variance in sample preparation (centre of diagram) and contribution of the LC-MS profiling to overall variance (left of diagram).
Figure 30.2.4
Figure 30.2.4
Graph of coefficients of variance (CV) versus peak integrated area for metabolites analyzed from pooled plasma using Basic Protocol 1. As the integrated peak area increases the CV decreases.
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