Rapid and efficient protein digestion using trypsin-coated magnetic nanoparticles under pressure cycles

Proteomics. 2011 Jan;11(2):309-18. doi: 10.1002/pmic.201000378. Epub 2010 Dec 17.


Trypsin-coated magnetic nanoparticles (EC-TR/NPs), prepared via a simple multilayer random crosslinking of the trypsin molecules onto magnetic nanoparticles, 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, whereas 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°C for 12 h or in combination with pressure cycling technology at room temperature for 1 min. In all cases, EC-TR/NPs performed equally to or better than free trypsin in terms of both the identified peptide/protein number and the digestion reproducibility. In addition, the concomitant use of EC-TR/NPs and pressure cycling technology resulted in very rapid (∼1 min) and efficient digestions with more reproducible digestion results.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Brain / metabolism
  • Enzymes, Immobilized / metabolism*
  • Magnetics*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Nanoparticles / chemistry*
  • Nanoparticles / ultrastructure
  • Pressure
  • Proteins / metabolism*
  • Proteome / metabolism
  • Proteomics / economics
  • Proteomics / methods*
  • Trypsin / metabolism*


  • Enzymes, Immobilized
  • Proteins
  • Proteome
  • Trypsin