19F NMR as a Tool for Monitoring Individual Differentially Labeled Proteins in Complex Mixtures.

Abstract

The ability to monitor the behavior of individual proteins in complex mixtures has many potential uses, ranging from analysis of protein interactions in highly concentrated solutions, modeling biological fluids or the intracellular environment, to optimizing biopharmaceutical co-formulations. Differential labeling NMR approaches, which traditionally use 15N or 13C isotope incorporation during recombinant expression, are not always practical in cases when endogenous proteins are obtained from an organism, or where the expression system does not allow for efficient labeling, especially for larger proteins. This study proposes differential labeling of proteins by covalent attachment of 19F groups with distinct chemical shifts, giving each protein a unique spectral signature which can be monitored by 19F NMR without signal overlap, even in complex mixtures, and without any interfering signals from the buffer or other unlabeled components. Parameters, such as signal intensities, translational diffusion coefficients, and transverse relaxation rates, which report on the behavior of individual proteins in the mixture, can be recorded even for proteins as large as antibodies at a wide range of concentrations.