Immobilization of trypsin onto multifunctional meso-fmacroporous core-shell microspheres: A new platform for rapid enzymatic digestion

A simple, fast, efficient, and reusable microwave-assisted tryptic digestion system which was constructed by immobilization of trypsin onto porous core-shell Fe304@fTiO2 microspheres has been developed. The nanostructure with magnetic core and titania shell has multiple pore sizes (2.4 and 15.0 nm), high pore volume (0.25 cm3 g-1), and large surface area (50.45 m2 g-1). For the proteins, the system can realize fast and efficient microwave-assisted tryptic digestion. Various standard proteins (e.g., cytochrome c (cytc), myoglobin (MYO), p-lactoglobulin (p-LG), and bovine serum albumin (BSA)) used can be digested in 45s under microwave radiation, and they can be confidently identified by mass spectrometry (MS) analysis; even the concentration of substrate is as low as 5 ng pi -1. Furthermore, the system for the 45s microwave-assisted tryptic digestion is still effective after the trypsin-immobilized microspheres have been reused for 5 times. Importantly, 1715 unique proteins from 10 tig mouse brain proteins can be identified with high confidence after treatment of 45 s microwave-assisted tryptic digestion. (C) 2014 Elsevier B.V. All rights reserved.