Development of Core-shell Magnetic Mesoporous SiO2 Microspheres for the Immobilization of Trypsin for Fast Protein Digestion

Abstract

In the work, we developed glycidoxypropyltrimethoxysilane (GLYMO)-modified Fe O @SiO core and perpendicularly aligned mesoporous SiO 2 shell (designated Fe O @nSiO @mSiO 2 ) as the novel substrate for the immobilization of large amount of trypsin and applied it for fast protein digestion.Firstly, Fe 3 O 4 @nSiO 2 @mSiO 2 microspheres were synthesized. Then, the surface of the microspheres was functionalized with GLYMO for enzyme immobilization.The amount of trypsin immobilized on was about 188 g/mg, which was much more than that on the previous magnetic materials. Using the trypsin-immobilized magnetic mesoporous SiO 2 microspheres, proteins in samples were fast digested with microwave irradiation. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analysis of the peptide fragmentation of three standard proteins, including cytochrome c (Cyt-c), myglobin (MYG), and bovine serum albumin (BSA). The functionalized magnetic microspheres served not only as substrate for enzyme immobilization, but also as excellent microwave absorbers, thus greatly improved the efficiency of protein digestion. It is also worth noting that by using this novel approach, the protein can be effectively digested within seconds, in contrast to hours required by conventional methods. Moreover, the trypsin-immobilized magnetic mesoporous SiO 2 microspheres exhibit better stability than conventional methods. Furthermore, the feasibility of using this novel strategy for real sample analysis was demonstrated by applying it to the analysis of human pituitary extraction which opens a route for its further application in large-scale proteomic analysis. Keywords: Mesoporous SiO 2 microspheres ; Peptide mapping analysis ; Microwave-assisted digestion ; MALDI-TOF MS IntroductionProteomic analysis of complex protein mixtures usually proceed along with either bottom-up or top-down approach. In both approaches, to obtain detailed structural information, proteins are selectively cleaved into smaller polypeptide fragments by controlled chemical or enzymatic reactions Wolters et al., 2001;Zhu et al., 2003 (Nalivaeva and Turner, 2001). The immobilized enzyme has been adopted to characterize the proteins with benefits from the reusability and stability of enzyme, the higher digestion efficiency of protein analytes, and no enzyme autolysis products (Dogruel et al., 1995;Nelson, 1997;Gobom et al., 1997; Jiang et al., 2000; Ekstrom et al., 2000;Peterson et al., 2002;Licklider et al., 1995;Ma et al., 2007;Svec, 2006). The main approach of enzyme immobilization is covalent binding. Epoxide is a classical tool for protein immobilization due to its versatile chemistry (Tischer and Wedekind, 1999; Petro et al., 1996). Petro and coworkers made the first attempt to immobilize trypsin on organic monoliths with epoxide groups in the late 1990s (Kvenková et al., 2005). And in many other reported approaches (Kvenková et al., 2005;Luo et al., 2002), the epoxide functional groups for enzyme immobilization are applied. As the auth...