Trypsin Immobilized on Magnetic Beads via Click Chemistry: Fast Proteolysis of Proteins in a Microbioreactor for MALDI-ToF-MS Peptide Analysis

Abstract

In the present work, magnetic poly(2-hydroxyethyl methacrylate–ethyleneglycol dimethacrylate) (mp(HEMA-EGDMA)) beads were prepared by suspension polymerization. The beads were grafted with poly(hydroxylpropyl methacrylate) (pHPMA) and used for immobilization of trypsin via click chemistry. The p(HPMA) units in the brushes made the fibrous polymer hydrophilic. Synthesized beads were characterized by swelling studies, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The bromo groups of the grafted polymer were modified into azide groups, and the enzyme was alkyne-functionalized for conjugation with azide-modified magnetic beads via click reaction. The kinetic constants (Km and Vmax) were determined by measuring initial reaction rates using the artificial chromogenic substrate N-benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA). The optimal pH value for the immobilized enzyme was slightly higher compared to free enzyme. The immobilized trypsin possessed much higher thermal stability than the free enzyme. Finally, the immobilized enzyme was placed in a microbioreactor for efficient proteolysis of model proteins, namely, bovine serum albumin (BSA), lysozyme, and cytochrome c. The immobilized trypsin in the microbioreactor provided a promising platform for model protein identification and was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) analysis.