Capillary Nanogel Separations for Biomolecular Separations Under Native Conditions

Capillary electrophoresis is a powerful separation technique for volume-limited samples in biomedical and pharmaceutical research. An advanced method is developed for native analyses using optical and mass spectrometry detection providing insight into protein structure and function. Protein-based assays are difficult to achieve with capillary electrophoresis under native conditions because of the interaction of proteins with the separation channel or the introduction of acidified solvents or organic additives. In this presentation new analytical technology is described that passivates the capillary surface with a semi-permanent coating. In addition, these capillary separations are enhanced by utilizing biocompatible nanogels, which have a thermally dependent viscosity. At temperatures below ~22°C nanogels have liquid-like viscosity. At higher temperatures nanogels have a gel-like viscosity. This property makes it easy to fill and pattern nanogels in narrow-bore capillaries at low temperatures using an automated capillary electrophoresis instrument. Once the nanogel is loaded into the capillary, the fluids are then locked in place by raising the temperature to gel the material. Proteins are separated under native conditions with nanogels. The migration time and resolution of the proteins increases with the viscosity of the nanogel. These separation additives are compatible with optical detection and with mass spectrometry when interfaced using vibrating sharp edge ionization driven by acoustic waves. With vibrating sharp-edge spray, the ionization is not dependent on pH, allowing the user to perform MS analyses in aqueous solutions at any desired pH. An approach for microscale separations of proteins under native conditions is described that leverages the low sample volume requirements of capillary electrophoresis. The technology outlined in this presentation provides new strategies to evaluate native protein structure and activity.