Core-shell silica microsphere-based trypsin nanoreactor for low molecular-weight proteome analysis

Core-shell mesoporous silica (CSMS) microspheres with tunable mesopores in the shell are highly desired in various bioapplications. With novel CSMS microspheres that are synthesized using a convenient two-phase process, we report in this study the analysis of low molecular-weight (MW < 30 kDa) proteins by combining size-exclusion separation and enzyme immobilization. The obtained CSMS microspheres possess uniform diameter (1.3 μm with a shell thickness of 57 nm), large and tunable perpendicular mesopores (7.9 nm), high surface area (55.5 m²/g), large pore volume (0.12 cm³/g) and excellent water dispersibility. The CSMS microsphere-based enzyme nanoreactors have been fabricated by immobilizing trypsin on the pore channels of the CSMS microspheres using either physical absorption or covalent binding via thiol or aldehyde group with a high loading capacity of 11.8-6.1 mg/g. Due to the unique fibrous pore structure, low MW proteins can enter the channels in the shell to interact with immobilized trypsin, followed by analysis of the digestion products using MALDI-TOF MS or electrophoresis (CE) techniques. The properties and analytical performance of different trypsin-immobilized CSMS microspheres has been systematically evaluated. The results show that the peptide-sequence coverage of the smaller protein is enhanced by using trypsin-CSMS microspheres, indicating the size-dependent digestion which results from the size-exclusion interaction of the mesopores against the high-MW proteins. The present study would pave the way for further applications of mesoporous materials in proteome analysis.