Assignment strategies for nuclear magnetic resonances in metabolomic research

Advances in nuclear magnetic resonance (NMR) spectroscopy over the past 3 decades have dramatically improved the sensitivity, spectral resolution, and versatility of NMR analysis, not only for macromolecular structural analysis but also for metabolite determination in complex biological mixtures (Fan, Higashi et al., 1986; Maaheimo, Fiaux et al., 2001; Emmerling, Dauner et al., 2002; Lindon, Holmes et al., 2004a, b; Fan and Lane 2008; Lane, Fan et al., 2008). It is now possible to determine metabolite structures present at submicromolar range in crude mixtures, a feat that was unthinkable less than a decade ago. NMR is one of the two major platforms for metabolomic analysis. Together with mass spectrometry (MS), the second platform, and chemometrics, it is now practical to profile an unprecedented number of metabolites in biological extracts or biofluids routinely (Fiehn, Kopka et al., 2000; Lutz 2005; Tohge, Nishiyama et al., 2005; Clayton, Lindon et al., 2006; Griffin, Blenkiron et al., 2006; De Vos, Moco et al., 2007; Fan and Lane 2008; Winder, Dunn et al., 2008; Fan, Yuan et al., 2010), which greatly facilitates the discovery of new functional markers of diseases or treatment responses. Both NMR and MS are even more powerful tools for mapping altered biochemical networks and regulation in organisms in response to perturbations owing to their ability to track isotopically enriched atoms in a large number of metabolites (Kikuchi, Shinozaki et al., 2004; Sherry, Jeffrey et al., 2004; Bian, Kasumov et al., 2005; Fan, Bandura et al., 2005; Fan and Lane 2008; Fan, Kucia et al., 2008; Lane, Fan et al., 2008; Fan, Lane et al., 2009; Lane, Fan et al., 2009; Fan 2010; Fan, Yuan et al., 2010; Fan and Lane 2011). In particular, multinuclear and multidimensional NMR techniques are uniquely suited for identifying and quantifying stable isotope labels at different atomic positions (i.e., positional isotopomers), which can be achieved without the need for authentic standards (Fan, Lane et al., 2001). This capability is unmatched by any other technique including MS.