In the work, we developed glycidoxypropyltrimethoxysilane (GLYMO)-modified Fe₃O₄@SiO₂ core and perpendicularly aligned mesoporous SiO₂ shell (designated Fe₃O₄@nSiO₂@mSiO₂) for the immobilization of trypsin for fast protein digestion.

Firstly, Fe₃O4@nSiO₂@mSiO₂ microspheres were synthesized. Then, the surface of the microspheres was functionalized with GLYMO for enzyme immobilization. Using the trypsin-immobilized magnetic mesoporous SiO₂ microspheres, proteins in samples were fast digested with microwave irradiation. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analysis of the peptide fragmentation of three standard proteins, including cytochrome c (Cyt-c), myglobin (MYG), and bovine serum albumin (BSA). The functionalized magnetic microspheres served not only as substrate for enzyme immobilization, but also as excellent microwave absorbers, thus greatly improved the efficiency of protein digestion. It is also worth noting that by using this novel approach, the protein can be effectively digested within seconds, in contrast to hours required by conventional methods. Moreover, the trypsin-immobilized magnetic mesoporous SiO₂ microspheres exhibit better stability than conventional methods. Furthermore, the feasibility of using this novel strategy for real sample analysis was demonstrated by applying it to the analysis of human pituitary extraction which opens a route for its further application in large-scale proteomic analysis.