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  • Title: Functionalized magnetic carbonaceous microspheres for trypsin immobilization and the application to fast proteolysis.
    Author: Yao G, Qi D, Deng C, Zhang X.
    Journal: J Chromatogr A; 2008 Dec 26; 1215(1-2):82-91. PubMed ID: 19026420.
    Abstract:
    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.
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