Immobilization of trypsin onto multifunctional meso-/macroporous 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 Fe 3 O 4 @fTiO 2 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   cm 3   g −1 ), and large surface area (50.45   m 2   g −1 ). For the proteins, the system can realize fast and efficient microwave-assisted tryptic digestion. Various standard proteins (e.g., cytochrome c (cyt-c), myoglobin (MYO), β-lactoglobulin (β-LG), and bovine serum albumin (BSA)) used can be digested in 45   s 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 μL −1 . Furthermore, the system for the 45   s microwave-assisted tryptic digestion is still effective after the trypsin-immobilized microspheres have been reused for 5 times. Importantly, 1715 unique proteins from 10   μg mouse brain proteins can be identified with high confidence after treatment of 45   s microwave-assisted tryptic digestion.