Fast Diffusion-Based Metabolomics Profiling by 3D Pure Shift HSQC-DOSY NMR with COLMARvista.

Peak overlap is a major obstacle for comprehensive metabolite profiling in NMR-based metabolomics. It is most severe for 1D 1H NMR spectra but can also persist for 2D NMR. Here, we demonstrate how metabolite-specific translational diffusion coefficients obtained from a pure shift pseudo-3D HSQC-DOSY experiment can resolve metabolite ambiguities caused by (near-)degenerate chemical shifts. In addition to the NMR pulse sequence, a novel pseudo-3D processing module implemented in COLMARvista is presented, which is a JavaScript-based standalone software for the automated processing, peak picking, quantification, and visualization of pseudo-3D HSQC-DOSY data sets. As is demonstrated for mouse urine, Pseudomonas aeruginosa biofilm, and a carbohydrate mixture, the pseudo-3D real-time pure shift HSQC-DOSY experiment is an effective tool for analyzing complex NMR metabolomics samples. Moreover, it is shown how the increased sensitivity and resolution of this experiment, compared to standard pseudo-3D-DOSY, yield more precise diffusion coefficients of the metabolomic mixture components even when using a minimal set of 2D planes.