Three-Dimensional Electrophoretic NMR Correlation Spectroscopy

A novel method of three-dimensional electrophoretic NMR correlation spectroscopy (3D EP-COSY) has been proposed, developed, and implemented. It has a demonstrated potential of facilitating simultaneous structural assignments of multiple proteins in mixtures. The principle is to add a pulsed DC electric field that introduces a new dimension of electrophoretic flow, in which resonances of different molecules can be separated by their electrophoretic migration rates without physical separation. As a result, two COSY spectra were simultaneously obtained in a single 3D EP-COSY experiment from a mixture of 150 mM l-aspartic acid and 148 mM 4,9-dioxa-1,12-dodecanediamine with concurrent resolution of their chemical shifts and J-coupling constants. This approach creates a new horizon of multidimensional electrophoretic NMR. The technical advance opens doors for structure characterization of complex protein systems and protein interactions, which are at the basis of biochemical mechanisms and the phenomena of living systems.