Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1215, Issue 1-2, Pages 82-91Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2008.10.114
Keywords
Magnetic carbonaceous microspheres; Immobilized trypsin; Microwave-assisted digestion; Proteomics; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Funding
- National Natural Science Foundation of China [20875017]
- National Basic Research Priorities Program [2007CB914100/3]
- Shanghai Leading Academic Discipline Project [B109]
- [PCRRF05001]
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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. (C) 2008 Elsevier B.V. All rights reserved.
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