Journal
PROTEOMICS
Volume 11, Issue 2, Pages 309-318Publisher
WILEY-BLACKWELL
DOI: 10.1002/pmic.201000378
Keywords
Enzyme coatings; Magnetic nanoparticles; Nanoproteomics; Pressure cycling technology; Protein digestion
Funding
- NIH National Center for Research Resources [RR018522]
- NIH National Cancer Institute [R21 CA12619-01]
- Pacific Northwest National Laboratory's (PNNL)
- Korean Ministry of Education, Science & Technology (MEST) [2009-0082314, 2009-0059861, 2009-0075638, FPR08A1-010]
- DOE [DE-AC05-76RLO1830]
- NATIONAL CENTER FOR RESEARCH RESOURCES [P41RR018522] Funding Source: NIH RePORTER
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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 degrees 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 (similar to 1 min) and efficient digestions with more reproducible digestion results.
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