Fabrication of trypsin-immobilized metal-organic framework-polymer monolithic spin column through a facile...

In this work, a novel trypsin-immobilized metal-organic framework (MOF)-polymer monolithic spin column was prepared by a facile biomineralization strategy, which intelligently utilizes the monolith to provide a porous support and the MOF to confer protective effect on trypsin. The proposed immobilized trypsin reactor showed higher enzyme-substrate affinity, better reusability, longer storage stability than the traditional covalently immobilized trypsin. Compared with free trypsin digestion, immobilization of trypsin by biomineralization exhibited enhanced pH and thermal stabilities with remarkably reduced digestion time, better economic performance due to reusability, without any loss of digestion activity. Finally, mouse liver proteomic analysis was carried out with emphasis on in-solution tryptic digestion and this immobilized trypsin spin column digestion, nearly twice the number of peptides and protein groups with varying molecular weights and isoelectric points were identified after spin column digestion within a much shorter hydrolysis time (from 14 h to just 1 h). Therefore, the biomineralized trypsin-MOF-monolithic spin column developed in our study appears to be an advanced tool in proteomic sample preparation, offering a combination of efficiency, speed, and robustness that is well-suited for both research and clinical areas.