Development of microwave-assisted protein digestion based on trypsin-immobilized magnetic microspheres for highly efficient proteolysis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

In this study, very easily prepared trypsin-immobilized magnetic microspheres were applied in microwave-assisted protein digestion and firstly applied for proteome analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Magnetic microspheres with small size were synthesized and modified by 3-glycidoxypropyltrimethoxysilane (GLYMO). Trypsin was immobilized onto magnetic microspheres through only a one-step reaction of its amine group with GLYMO. When these easily prepared trypsin-immobilized magnetic microspheres were applied in microwave-assisted protein digestion, the magnetic microspheres not only functionalized as substrate for trypsin immobilization, but also as an excellent microwave absorber and thus improved the efficiency of microwave-assisted digestion greatly. Cytochrome c was used as a model protein to verify its digestion efficiency. Without any additives such as organic solvents or urea, peptide fragments produced in 15 s could be confidently identified by MALDI-TOF-MS and better digestion efficiency was obtained comparing to conventional in-solution digestion (12 h). Besides, with an external magnet, trypsin could be used repeatedly and at the same time no contaminants were introduced into the sample solution. It was verified that the enzyme maintained high activity after seven runs. Furthermore, reversed-phase liquid chromatography (RPLC) fractions of rat liver extract were also successfully processed using this novel method. These results indicated that this fast and efficient digestion method, which combined the advantages of immobilized trypsin and microwave-assisted protein digestion, will greatly hasten the application of top-down proteomic techniques for large-scale analysis in biological and clinical research. Copyright 2007 John Wiley & Sons, Ltd.