Mechanism exploration of adsorption-immobilized enzymatic reactor using polymer-coated silica microbeads

A verified mechanism of adsorption-immobilized enzymatic reactor for enhanced proteolysis is presented. Silica microbeads coated with poly (diallyldimethylammonium chloride) (PDDA) or poly (styrene sulfonate) (PSS) were used to trap trypsin and proteins on the surface through electrostatic interactions in order to improve digestion efficiency. Charge states measured by zeta-potentials showed their positively and negatively charged respectively. We found that high proteolytic efficiency could be achieved only if both proteases and proteins were adsorbed by materials. Once the proteins and proteases were confined together in a nanoscopic area, the enrichment of the substrate could lead to a high performance proteolytic effect. Electrostatic interactions were considered as the predominant adsorption factor rather than hydrophilic/hydrophobic interactions. In less than 5 min, in the presence of PSS-coated silica beads, 10 peptides digested from positively-charged cytochrome C were detected by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF), with the high sequence coverage up to 63%, while using PDDA-coated silica beads or conventional in-solution digestion yielded only 5 detectable peptides and 39% sequence coverage was obtained. Ovalbumin seemed incompatible with any kind of charged-material-aided tryptic digestion. The mechanism of adsorption-immobilized enzymatic processes has also been studied in detail. The adsorption equilibrium was proven to be attained in less than one minute, and the proteolytic procedure was regarded as the rate-determining step. This study provides a reasonable mechanism for an adsorption-material catalyzed proteolytic procedure and a promising guideline for designing the next generation of high-performance enzymatic reactors.