Efforts to determine the temporal concentrations of the vast number of proteins in a cell (proteomics) are placing unprecedented demands on techniques for protein separation and analysis. Additionally, the development of recombinant proteins for both research and therapeutic applications requires efficient methods for protein purification. This dissertation describes the development of two kinds of materials for protein separation and analysis: polymer brush-modified magnetic nanoparticles and trypsin-modified membranes.The growth of poly(2-hydroxyethyl methacrylate) brushes on magnetic nanoparticles and subsequent brush functionalization with nitrilotriacetate-Ni2+ yield magnetic beads that selectively capture polyhistidine-tagged (His-tagged) protein directly from cell extracts. Transmission electron microscopy, FT-IR spectroscopy, thermogravimetric analysis, and magnetization measurements confirm and quantify the formation of the brushes on magnetic particles, and multilayer protein adsorption to these brushes results in binding capacities (220 mg protein/g of beads) that are an order of magnitude higher than those of commercial magnetic beads. Moreover, the functionalized beads selectively capture His-tagged protein within 5 min. The high binding capacity, high protein purity, and short incubation time make brush-modified particles attractive for purification of recombinant proteins. Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is much higher than the 49% coverage obtained with in-solution digestion for 16 h or the sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt% sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra, even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.I also present a preliminary investigation of how the number and size of peptide fragments vary with the protein residence time in the trypsin-containing membranes. We hope to use extremely short residence time to create large protein segments for potential applications in middle-down proteomics, where fragmentation of modest sized (3-20 kDa) protein pieces in the mass spectrometer facilitates protein sequencing and discovery of post-translational modifications.
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