Hollow silica bubble based immobilized trypsin for highly efficient proteome digestion and buoyant separation

Abstract

Tryptic digestion before identification and quantification by mass spectrometry is an indispensable process for most proteomics studies. Conventional in-solution digestion process always suffers from incomplete digestion and is time-consuming and expensive. Development of a novel proteolytic digestion method with fast, high efficiency and reusability of the enzyme is still an urgent task. In this study, new hollow silica bubbles-based immobilized trypsin (trypsin@SiB) was developed via brushes structured glycidyl methacrylate (GMA) grafted on the silica bubbles (SiB) by the reversible addition–fragmentation chain transfer polymerization (RAFT) technique followed by trypsin's immobilization on the microsphere. The new immobilized trypsin not only showed high efficiency and stability but also reusability. Due to the low density (0.6 g cm⁻³) of SiB, it can float over the solution for several minutes so that the immobilized trypsin can be separated and recovered from the system easily. Highly efficient digestion of BSA was achieved with this trypsin@SiB within 1 min and the obtained sequence coverage (92%) was better than that from conventional in-solution digestion for more than 12 h (76%). To further confirm the efficiency of trypsin@SiB for complex proteomic analysis, the protein extracted from human urine was analyzed as a real sample. Within 10 min digestion, 510 protein groups were identified with the LC-MS/MS analysis and database searching, whereas the number of identified proteins after 12 h in-solution digestion was 493 with the same identification conditions. The successful application of trypsin@SiB demonstrated its potential as a high efficient digestion method for future proteomics analysis.