In the work, we developed glycidoxypropyltrimethoxysilane (GLYMO)-modified Fe3O4@SiO2 core and perp
moreIn the work, we developed glycidoxypropyltrimethoxysilane (GLYMO)-modified Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2 shell (designated Fe3O4@nSiO2@mSiO2) as the novel substrate forthe immobilization of large amount of trypsin and applied it for fast protein digestion.Firstly, Fe3O4@nSiO2@mSiO2 microspheres were synthesized. Then, the surface of the microspheres was functionalized with GLYMO for enzyme immobilization.The amount of trypsin immobilized on Fe3O4@nSiO2@mSiO2 was about 188 ì g/mg, which was much more than that on the previous magnetic materials. 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 massspectral 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.
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