High-resolution electrophoretic separations of native state proteins can provide a wealth of information beyond molecular weight determinations. Preserving native structures enables studies of protein folding states, association of protein complexes, ligand binding, and post-translational modifications. Such information cannot be determined by standard denaturing polyacrylamide gel electrophoresis. However, native polyacrylamide gel electrophoresis suffers from limited separation resolution and lengthy analysis times. This has led many researchers to forgo more information-rich native analyses in favor of more robust denatured analyses. This project seeks to bridge the gap between the high quantity of information provided by native gels and the high quality of data provided by denatured gels. We will develop a robust microfluidic gel electrophoresis platform to comprehensively characterize native proteins. An innovative strategy employing a thermally reversible gel will be evaluated to generate high-resolution separations. Introduction of temperature as an additional adjustable parameter will enable separation performance to be tuned on-demand while preserving native protein structures.
The specific aims of this proposal will establish two core capabilities necessary to demonstrate the suitability of the proposed approach for native protein characterizations. The overall mass range of the system will be characterized in Aim 1 to maximize dynamic range by evaluating thermal gel compositions over a range of temperatures.
Aim 2 will implement thermal gradients for fine control over resolution to separate different folding states of a single protein. Milestones have been defined to describe expected performance capabilities resulting from each aim, building towards the development of a flexible electrophoresis platform. Ultimately, the proposed system is anticipated to surpass performance of current native protein characterization methods and accommodate diverse biochemical research applications including analysis of multimeric protein complexes and evaluating stability of proteins and antibodies in biotherapeutic formulations.
Standard gel electrophoresis methods preclude biophysical characterizations of proteins because samples are denatured prior to analysis, which obscures information on conformation states or association of biocomplexes. This project will develop a versatile microfluidic gel electrophoresis platform to tune separation resolution of native proteins on-demand. The proposed system will integrate numerous characterizations into a single, information-rich analysis to facilitate broad biomedical and pharmaceutical research applications.
