Enzyme Coatings On Magnetic Nanoparticles for Rapid Protein Digestion In Proteomic Analysis

Protein digestion is a critical step in the bottom-up proteomic analysis, but takes a long time due to the poor stability of a digestive enzyme called trypsin. We prepared trypsin coatings on magnetic nanoparticles (EC-TR/NPs) via a simple multilayer random crosslinking of the trypsin molecules onto magnetic nanoparticles. The resulting EC-TR/NPs were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, while the conventional immobilization of covalently-attached trypsin on magnetic nanoparticles resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. A single model protein, a five protein mixture, and a whole mouse brain proteome were digested at traditional digestion condition or in combination with pressure cycling technology (PCT) at room temperature for 1 min. Highly stable and magnetically separable EC-TR/NPs were successfully employed in protein digestion for proteomic analysis. In addition, the concomitant use of EC-TR/NPs and PCT resulted in very rapid (~1 min) and efficient digestions with more reproducible digestion results.