Functionalized magnetic carbonaceous microspheres for trypsin immobilization and the application to fast proteolysis

In this study, magnetic carbonaceous (MC) microspheres prepared with a large-scale synthesis approach were developed as the novel substrate for enzyme immobilization, and the trypsin-immobilized MC microspheres were successfully applied to protein fast digestion. Firstly, MC microspheres with small size, strong magnetism, and biological compatibility were prepared through two-step solvothermal reactions. Secondly, MC microsphere surface was modified by 3-glycidoxypropyltrimethoxysilane (GLYMO). Finally, the enzyme was immobilized on the GLYMO-functionalized MC microspheres. The enzyme-immobilized magnetic microspheres were applied for fast protein digestion with microwave-assistance. Bovine serum albumin, myoglobin and cytochrome c, were used as model proteins to verify the digestion efficiency, and the digestion products were then characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with sequence coverage of 43%, 90% and 77%, respectively. The enzyme-immobilized magnetic particles were also successfully applied to the analysis of human pituitary extract. After database search, 485 proteins (p<0.01) were identified when the extract was digested by the microspheres. This opens a route for its future application in bottom-up proteomic analysis.