Rapid Protein Digestion based on Trypsin Coated Magnetic Nanoparticles under Pressure Cycles

Ttrypsin digestion is a critical step in the bottom-up proteomic analysis, but takes a long time due to the poor stability of trypsin itself. We prepared trypsin-coated 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 NPs 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 atmospheric pressure and 37 oC for 12 h or in combination with pressure cycling technology (PCT) at room temperature for 1 min. In all cases, EC-TR/NPs performed equally to or better than free trypsin in terms of both identified peptide/protein number and the digestion reproducibility. 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.