Rapid and Efficient Proteolysis for Protein Analysis by an Aptamer-Based Immobilized Chymotrypsin Microreactor

Abstract

Efficient protein digestion is a key step for successful mass spectrometry identification. However, traditional in-solution digestion suffers some drawbacks, such as autolysis of protease, long analysis times and lack of control. Recently, specific single-stranded nucleic acids, aptamers, screened from random sequence pools, have been performed high affinity for targets. In this paper, we have developed a novel enzyme reactor, which immobilized chymotrypsin based on aptamer-grafted silica beads. Mixed proteins, which consist of bovine serum albumin, myoglobin, and cytochrome c, were used as samples, to evaluate the digestion performance of the enzymatic reactor. With the use of this novel tool, proteins were digested in 40 min to an extent similar to that achieved with soluble enzyme at 37 C after 16 h. Moreover, enzymatic reactor regeneration was carried out through chymotrypsin elution and re-immobilization. The advanced characteristics of the aptamer-based chymotrypsin reactor demonstrated that aptamers could serve as novel materials for rapid and efficient enzyme immobilization and application in protein studies.

Preparation of trypsin aptamer modified silica particles by surface initiated atom transfer radical polymerization for proteome identification

“…9 Xiao Peng et al (in our group) obtained chymotrypsin aptamers by systemic evolution of ligands by exponential enrichment (SELEX) technology and an aptamerbased immobilized chymotrypsin microreactor was developed exhibiting excellent enzymatic capacity. 10 Moreover, in our previous work, an aptamer for trypsin was selected and characterized in vitro. The aptamer-based trypsin reactor was developed for proteomic digestion using glutaraldehyde as a linker.…”