title: Characterization of efficient proteolysis by trypsin loaded macroporous silica.
abstract: Tryptic digestion of proteins in trypsin loaded porous silica has been shown to be highly efficient. Enzymatic silica-reactors were prepared by immobilizing trypsin into macroporous ordered siliceous foam (MOSF) and into mesoporous SBA-15 silica which has a smaller pore size. The tryptic products from the silica reactors were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a higher proteolysis efficiency was obtained with MOSF. These results can be well interpreted by a sequential digestion model taking into account the confinement and concentration enrichment of both the substrates and enzymes within the silica pores. Proteins at low concentrations and proteins in urea and surfactant solutions were also successfully digested with the MOSF-based reactor and identified by MS. Considering that the immobilized trypsin could retain its enzymatic activity for weeks, this MOSF reactor provides many advantages compared to free enzyme proteolysis. As a proof-of-concept, the digest of a real complex sample extracted from the cytoplasm of mouse liver tissue using trypsin loaded MOSF yielded better results than the typical in-solution protocol.
authors.affiliation: Department of Chemistry, Fudan University, Shanghai, People's Republic of China.
authors.forename: Weichao
authors.initials: W
authors.lastname: Guo
authors.forename: Hongyan
authors.initials: H
authors.lastname: Bi
authors.forename: Liang
authors.initials: L
authors.lastname: Qiao
authors.forename: Jingjing
authors.initials: J
authors.lastname: Wan
authors.forename: Kun
authors.initials: K
authors.lastname: Qian
authors.forename: Hubert H
authors.initials: HH
authors.lastname: Girault
authors.forename: Baohong
authors.initials: B
authors.lastname: Liu
mesh_terms: D059748:Proteolysis; D012822:Silicon Dioxide / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry*
chemical_list: D012822:Silicon Dioxide; D014357:Trypsin