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

•A novel microwave-assisted tryptic digestion system.•Characterization with SEM, TEM, EDX, XRD, and FT-IR.•The meso-/macroporous shell structure withthe high affinity and loading capacity of trypsin.•Decrease of digestion time up to less than 1 min.•MALDI-MS and nanoLC-MS analysis with database identification.

A simple, fast, efficient, and reusable microwave-assisted tryptic digestion system which was constructed by immobilization of trypsin onto porous core-shell Fe3O4@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 (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.

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