Applications of NMR spectroscopy to systems biochemistry

Teresa W.-M. Fan, Andrew N. Lane
2016 Progress in nuclear magnetic resonance spectroscopy  
The past decades of advancements in NMR have made it a very powerful tool for metabolic research. Despite its limitations in sensitivity relative to mass spectrometric techniques, NMR has a number of unparalleled advantages for metabolic studies, most notably the rigor and versatility in structure elucidation, isotope-filtered selection of molecules, and analysis of positional isotopomer distributions in complex mixtures afforded by multinuclear and multidimensional experiments. In addition,
more » ... has the capacity for spatially selective in vivo imaging and dynamical analysis of metabolism in tissues of living organisms. In conjunction with the use of stable isotope tracers, NMR is a method of choice for exploring the dynamics and compartmentation of metabolic pathways and networks, for which our current understanding is grossly insufficient. In this review, we describe how various direct and isotope-edited 1D and 2D NMR methods can be employed to profile metabolites and their isotopomer distributions by stable isotope-resolved metabolomic (SIRM) analysis. We also highlight the importance of sample preparation methods including rapid cryoquenching, efficient extraction, and chemoselective derivatization to facilitate robust and reproducible NMR-based metabolomic analysis. We further illustrate how NMR has been applied in vitro, ex vivo, or in vivo in various stable isotope tracerbased metabolic studies, to gain systematic and novel metabolic insights in different biological systems, including human subjects. The pathway and network knowledge generated from NMRand MS-based tracing of isotopically enriched substrates will be invaluable for directing functional analysis of other 'omics data to achieve understanding of regulation of biochemical systems, as demonstrated in a case study. Future developments in NMR technologies and reagents to enhance both detection sensitivity and resolution should further empower NMR in systems biochemical research. The assignment of known metabolites in complex mixtures by NMR can be based on a number of NMR parameters, including chemical shifts and their pH dependence, spin multiplicity, homonuclear and heteronuclear covalent connectivity, and the nuclear Overhauser effect. These aspects have been reviewed by us previously and will not be elaborated here [22, 23] . The past decade of development in metabolite databases and database search tools has made it practical to assign metabolites by comparing the NMR spectral features of given samples against those of standards compiled in databases. There are now quite a few public /index.php/toccata2/index) [24, 25] and proprietary (e.g. ChenomX, Bruker) databases available for metabolite assignment purpose. As NMR parameters can be sensitive to pH, temperature, concentration, and ionic conditions [26, 27] , the sample spectra will need to be acquired under matched conditions with the standard spectra acquired in the databases. This is a crucial aspect of NMR database search for metabolite assignment, since many laboratories differ in their extraction protocols. In addition, at some pH values, the chemical shifts can be degenerate for common metabolites, such as lactate and threonine at neutral pH [26] . Furthermore, 1D databases that rely primarily on chemical shifts can be unreliable and only a limited number of features are identified, due to spectral crowding and lack of more rigorous indication of structural features such as covalently linked partners. For Fan and Lane
doi:10.1016/j.pnmrs.2016.01.005 pmid:26952191 pmcid:PMC4850081 fatcat:km3sopobargb5hm35teonftmlq