title: Rapid and efficient protein digestion using trypsin-coated magnetic nanoparticles under pressure cycles.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, Korea University, Seoul, Korea.
authors.forename: Byoungsoo
authors.initials: B
authors.lastname: Lee
authors.forename: Daniel
authors.initials: D
authors.lastname: Lopez-Ferrer
authors.forename: Byoung Chan
authors.initials: BC
authors.lastname: Kim
authors.forename: Hyon Bin
authors.initials: HB
authors.lastname: Na
authors.forename: Yong Il
authors.initials: YI
authors.lastname: Park
authors.forename: Karl K
authors.initials: KK
authors.lastname: Weitz
authors.forename: Marvin G
authors.initials: MG
authors.lastname: Warner
authors.forename: Taeghwan
authors.initials: T
authors.lastname: Hyeon
authors.forename: Sang-Won
authors.initials: SW
authors.lastname: Lee
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Jungbae
authors.initials: J
authors.lastname: Kim
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism*; D008280:Magnetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D011312:Pressure; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000191:economics / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D011506:Proteins; D020543:Proteome; D014357:Trypsin