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Rapid and efficient protein digestion using trypsin‐coated magnetic nanoparticles under pressure cycles
Author(s) -
Lee Byoungsoo,
LopezFerrer Daniel,
Kim Byoung Chan,
Na Hyon Bin,
Park Yong Il,
Weitz Karl K.,
Warner Marvin G.,
Hyeon Taeghwan,
Lee SangWon,
Smith Richard D.,
Kim Jungbae
Publication year - 2010
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000378
Subject(s) - trypsin , autolysis (biology) , chemistry , chromatography , digestion (alchemy) , denaturation (fissile materials) , nanoparticle , covalent bond , magnetic nanoparticles , peptide , biochemistry , enzyme , materials science , nanotechnology , nuclear chemistry , organic chemistry
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.