| Abstract
| - Various silica-based microreactors have been designedthat use enzyme immobilization to address technicalconcerns in proteolysis including inefficient and incomplete protein digestion. Most of current designs for proteolytic reactors can improve either protease stability orproteolysis efficiency of individual protein(s). However,the desired features such as rapid digestion, largersequence coverage, and high sensitivity have not beenachieved by a single microreactor design for broad rangeproteins with diverse physical properties. Here, unlikeconventional enzyme immobilization strategies, we describe a novel proteolytic nanoreactor based on the uniquethree-dimensional nanopore structure of our newly synthesized mesoporous silica (MPS), FDU-12, which integrates substrate enrichment, “reagent-free” protein denaturation, and efficient proteolytic digestion. In ourdesign, protein substrates were first captured by MPSnanopore structure and were concentrated from thesolution. Following the pH change and applying trypsin,the denaturation and concurrent proteolysis of broad-range proteins were efficiently achieved. In minutes, manymore sample peptides from the in-nanopore digestion ofprotein mixtures were detected by mass spectrometry,resulting in the identifications of a broad range of diverseproteins with high sequence coverage. The unique features of FDU-12 nanostructure that allow rapid, completeproteolysis and resulting enhanced sequence coverage ofindividual proteins were investigated by using Ramanspectroscopy and comparative studies with respect toother MPSs.
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