Characterization of protein mixtures by ion-exchange chromatography coupled on-line to nuclear magnetic resonance spectroscopy

Abstract The first example of HPLC–NMR analysis of proteins is reported. By this means, a mixture of hen egg lysozyme and horse heart cytochrome was separated and identified. The chromatographic separation is based on ion-exchange HPLC, which is likewise for the first time coupled to 1 H NMR. In the stop-flow mode, characteristic one-dimensional 1 H spectra, two-dimensional total correlation data sets (TOCSY), and nuclear overhauser effect correlation spectra (NOESY) of the two proteins were obtained. In addition, lysozyme from crude hen egg white was unambiguously characterized by using a stop-flow HPLC–NMR-TOCSY experiment. These experiments extend the applicability of HPLC–NMR to the rapid analysis of protein mixtures.