ABSTRACTFUNCTIONALIZED POROUS MEMBRANES FOR RAPID PROTEIN PURIFICATION AND CONTROLLED PROTEIN DIGESTION PRIOR TO MASS SPECTROMETRY ANALYSISByJinlan DongWith the increasing demand for proteins in biotechnological research and applications, high-throughput protein purification is crucial. Convective mass transport and short radial diffusion distances in membrane pores make membrane absorbers attractive for rapid protein capture. However, the binding capacity of membranes is modest compared to bead-based techniques. Layer-by-layer polyelectrolyte adsorption should provide a simple way to functionalize membranes for extensive protein capture. Preliminary studies showed that poly(allylamine) (PAH)/poly(acrylic acid) (PAA) films adsorbed at pH 3 on Au-coated wafers swell up to 500% in pH 7 buffer. At neutral pH, (PAH/PAA)n films are highly ionized and capture the equivalent of many monolayers of lysozyme without causing changes in film roughness. Nylon membranes containing PAA/polyethyleneimine (PEI)/PAA films adsorbed at pH 3 provide a porous platform with a high density of –COOH groups that are available for further derivatization with NTA-Ni2+ complexes. When modified with PAA/PEI/PAA-NTA-Ni2+, membranes enable rapid isolation of His-tagged proteins from cell extracts with > 95% purity and recovery. Amazingly, the whole protein purification takes less than 30 min. A new membrane device developed by our commercial partners should require only 5 min to purify His-tagged protein from cell extracts. Finally, a polymer containing NTA as part of its side chain will avoid derivatization steps and greatly simplify the membrane-modification process. Membrane adsorbers can also include immobilized proteins as their affinity agents. However, a membrane containing Concanavalin A (Con A, a protein that binds specific glycoproteins) did not provide a high glycoprotein binding capacity. Nevertheless, an antibody-containing membrane has the potential for rapid immunoaffinity purification of specific antigens with a binding capacity significantly higher than that of commercial resins. Thin membranes also present an excellent platform for creating enzyme reactors. Covalent binding to PAA-modified membranes immobilizes about 35 mg of trypsin per cm3 of membrane pore, which is a 2800-fold increase in enzyme concentration relative to in-solution digestion. This high enzyme concentration also yields a 75-fold increase in digestion rate compared to typical in-solution methods. In addition, the covalent immobilization successfully prevents enzyme leaching and stabilizes the enzyme in 6 M urea or at pH 5, which aids digestion of proteins that require denaturation. Even after 6 months of storage at room temperature, the membrane can still digest protein in ms residence times. Moreover, thin (110 μm) membrane reactors afford fine control over proteolysis time and, hence, peptide lengths to enhance protein analysis. For β-casein digestion, msec digestion gives a large peptide that carries all five phosphorylation sites. Thus, such large peptides should enable correlation of post-translational protein modification events such as phosphorylation.
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