Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith.

A metal-ion chelate immobilized enzyme reactor (IMER) supported on organic-inorganic hybrid silica monolith was developed for rapid digestion of proteins. The monolithic support was in situ prepared in a fused silica capillary via the polycondensation between tetraethoxysilane hydrolytic sol and iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane. After activated by Cu(2+) , trypsin was immobilized onto the monolithic support via metal chelation. Proteolytic capability of such an IMER was evaluated by the digestion of myoglobin and BSA, and the digests were further analyzed by microflow reversed-phase liquid chromatography with ESI-MS/MS. Similar sequence coverages of myoglobin and BSA were obtained by IMER, in comparison to those obtained by in-solution digestion (91 versus 92% for 200 ng myoglobin, and 26 versus 26% for 200 ng BSA). However, the digestion time was shortened from 12 h to 50 s. When the enzymatic activity was decreased after seven runs, the IMER could be easily regenerated by removing Cu(2+) via EDTA followed by trypsin immobilization with fresh Cu(2+) introduced, yielding the equal sequence coverage (26% for 200 ng BSA). For ∼5 μg rat liver extract, even more proteins were identified with the immobilized trypsin digestion within 150 s in comparison to the in-solution digestion for 24 h (541 versus 483), demonstrating that the IMER could be a promising tool for efficient and high-throughput proteome profiling.