Efficient protein digestion using immobilized trypsin onto tannin modified Fe₃O₄ magnetic nanoparticles

Fe₃O₄ magnetic nanoparticles (MNPs) were prepared via solvothermal method. A commercially available trypsin was covalently immobilized onto MNPs modified with tannin (T) via a novel binding process. The morphology, structure, surface and magnetic properties of the obtained nanostructures were characterized comprehensively. The Fe₃O₄ MNPs had a saturation magnetization value of 60.18emu/g at room temperature, while the tannin modified Fe₃O₄ MNPs, and the trypsin immobilized on tannin modified-Fe₃O₄ MNPs possessed a saturation magnetization value of 57.82emu/g and 55.16emu/g, respectively, which indicated the decent tannin coating and trypsin immobilization. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of widely used bovine serum albumin (BSA). The immobilized trypsin was investigated by conducting the tryptic digestion of BSA within 1min, 5min and 15min. Also, microwave-assisted digestion was carried out for 15s. The digested protein fragments were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and a satisfactory peptide numbers of 39, and a superior sequence coverage of 84% for 1min digestion were obtained. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed the satisfactory digestion of BSA and egg white proteins by immobilized trypsin.