Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

Abstract

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Membrane and Bioseparation

“…In the Figure 6. Schematic diagram of (a) growth of polymer brushes and (b) layer-by-layer adsorption to form films that may capture proteins in membrane pores [65]. "grafting-to" method, the limitations of surface access for the incoming polymeric chains are referred to relatively low thickness and bond densities; in contrast, the "grafting-from" method uses small monomers, which, to provide relatively high bond densities, easily reach the surface growing reactive [11].…”

“…Several groups modified a variety of membranes using ATRP from immobilized initiators, and binding capacities of such membranes often exceed 100 mg of protein per milliliter of membrane [42,[80][81][82][83]. The amount of protein binding in polymer brushes varies with polymer-chain areal density; low-chain densities yield few binding sites and minimal protein capture, whereas high densities may result in steric hindrance to protein entry into the brush [65]. Hence, an intermediate chain areal density will likely lead to the most protein binding [65].…”

“…The amount of protein binding in polymer brushes varies with polymer-chain areal density; low-chain densities yield few binding sites and minimal protein capture, whereas high densities may result in steric hindrance to protein entry into the brush [65]. Hence, an intermediate chain areal density will likely lead to the most protein binding [65]. Chain density depends in part on the density of initiation sites anchored to membrane surfaces, and anchoring typically occurs through surface functionalities such as hydroxyl groups [84] and carboxylic acids [85].…”

“…Adsorption of proteins depends on the surface charge, protein charge, and the thickness of the polyelectrolyte film [175]. Generally, protein binding in LBL films depends on the size of the membrane pore, hydrophobicity, and surface charge [65]. DOI: http://dx.doi.org /10.5772/intechopen.86954 Membranes containing film PAA/BPEI/PAA bind 100 mg/ml lysozyme through ion exchange [11], which is about twice the capacity binding of the commercial ion exchange membranes.…”

“…When the protein is captured from the cell extract, the size of the pores greater than 1 μm prevents the blocking of pores, and to maintain the flow speed is important [65].…”