CAREER: Electrophoresis of Proteins in Templated Nanoporous Materials

Princeton University, Princeton NJ

Investigators

Abstract

CAREER: ELECTROPHORESIS OF PROTEINS IN TEMPLATED NANOPOROUS MATERIALS This CAREER project brings together biophysical and bioanalytical chemistry, with materials science and nanotechnology, to address challenges in the field of proteomics. The description of complex mixtures of proteins that make up a cell is done through a combination of separation and identification. Separations are almost always done under conditions that denature proteins and thereby obliterate their function. We seek new materials that will support the rapid and efficient separation of complex mixtures of proteins under conditions that maintain native protein structure and function. Proteins separated in these materials will be available immediately for the screening of function, such as ligand binding or enzymatic activity. We propose to develop nanoporous silica, produced using surfactant liquid-crystal templates, as a new matrix material for the electrophoretic separation of proteins under non-denaturing conditions. These materials will be fabricated into thin-films and microchannel arrays. Models of the electrophoretic motion of proteins in nanochannels should guide identification of optimal separation conditions. Our fundamental understanding of this process, however, is incomplete. Protein charge ladders - collections of protein derivatives that differ incrementally in net charge - have been useful in testing models of protein electrophoresis in free solutions. We will use charge ladders to study the role of protein charge and hydrodynamic size on the efficiency of separation and resolution of different proteins in nanochannels. Knowledge gained from these fundamental studies will test current models of electrophoresis in nanochannels and direct the formulation of new models that will, in turn, aid in the development of optimal conditions for protein separations. Proteomics - the study of the complete protein complement to the genome - seeks to answer the following two questions. Which proteins are expressed (i.e., are present and functional) in a cell? What are the functional interactions between the expressed proteins and other molecules in the cell? Answers to the first question require the separation and identification of complex mixtures of proteins isolated from cells. Separations are usually done under conditions that denature proteins and thereby obliterate their function. Answers to the second question require the biochemical analysis of individual proteins; analysis is usually done with isolated samples of pure proteins in their native functional state. Current attempts to answer these two questions therefore involve independent and mutually exclusive techniques. Our approach would combine separation with analysis of protein function. The goal is to integrate protein separation, identification and analysis of function on a single "chip". This integrated approach to protein analysis requires new matrix materials that allow separations of proteins under conditions that maintain their native structure and function. We believe our approach will allow this integration. The development of these templated silica materials and the understanding of the transport of proteins through them via electrophoresis should also have significant impact on the use of such materials in bioprocess engineering. This CAREER award integrates research with an educational program that includes new course development at the university level, research opportunities for high school students, and focused educational outreach to elementary schools. The central theme of this educational program is that engineering applications and technology are effective "hooks" that introduce and draw students into the basic sciences and mathematics.