Characterizing the Spin System Using 3D TOCSY J-Resolved Spectroscopy to Facilitate Deep Annotation of Individual Peaks within Interleaved Multiplets in High-Resolution 1D ¹H Spectra of Cellular Metabolic Mixtures

One-dimensional (1D) ¹H nuclear magnetic resonance (NMR) is often employed in metabolomics studies. In this study, 3D TOCSY J-resolved spectroscopy has been proposed to facilitate the characterization of a spin system with multiple chemical shifts, multiplicities, and J coupling constants. The combined information on the whole spin system allows for unambiguous annotation of individual peaks, including those within interleaved or closely spaced multiplets in a high-resolution 1D ¹H NMR spectrum using the complete set of chemical shifts, multiplicities, and J couplings from an NMR database as a distinct identifier for the metabolite. The method was demonstrated on a simple model mixture comprising five human metabolite standards and a complex cellular metabolic mixture. It facilitated deep annotation of 242 characteristic peaks in the high-resolution 1D ¹H NMR spectrum and enabled unambiguous identification of 51 metabolites within the complex cellular metabolic mixture.