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.

Keywords:

• Aptamer
• Chymotrypsin
• IMER
• Protein identification

Acknowledgments

P. Xiao and X. Lv contributed equally to this work.

We would like to acknowledge the financial supports from The National Key Technology R&D Program of China (Grant No. 2009BAK59B01) and The National Nature Science Foundation of China (Grant No. 21275019).

Notes

^a Values are averages from five consecutive experiments.

^a Valuesare averages from three consecutive experiments.