Trypsin Immobilization on Hairy Polymer Chains Hybrid Magnetic Nanoparticles for Ultra Fast, Highly Efficient Proteome Digestion, Facile 18O Labeling and Absolute Protein Quantification

In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile O-18 labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using O-18 labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification.