Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers
- Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers
- 이병수; 김병찬; 장문석; 김한솔; 나현빈; 박용일; 이진우; 현택환; 이후근; 이상원; 김중배
- Trypsin digestion; Nanobiocatalysis
- Issue Date
- Chemical engineering journal
- VOL 288, 770-777
- Protein digestion, using an enzyme called trypsin (TR), is one of the key steps in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and not-automated due to the poor stability and autolysis of trypsin. To improve the protein digestion process, trypsin was immobilized and stabilized on polymer nanofibers entrapping superparamagnetic nanoparticles (magnetic nanofibers, NP-NFs). By electrospinning the homogeneous mixture of superparamagnetic nanoparticles (NPs) and polystyrene-poly(styrene-co-maleic anhydride), NPs could be effectively entrapped within polymer nanofibers, generating magnetically-separable nanofibers with high surface area for trypsin immobilization via the approach of enzyme coatings. Trypsin coatings on magnetic nanofibers (EC-TR/NP-NFs; EC-TR), fabricated via simple attachment of crosslinked trypsin molecules onto NP-NFs, were highly stable and could be recycled via facile magnetic separation. EC-TR showed negligible loss of trypsin activity even after incubation in an aqueous buffer under rigorous shaking (200 rpm) for 80 days, while the control samples of covalently-attached trypsin on NP-NFs (CA-TR/NP-NFs; CA-TR) and free trypsin lost more than 90% of their initial activities within 11 and 6 days, respectively. When highly-stable and magnetically-separable EC-TR was employed for the repetitive digestions of enolase under recycled uses for the duration of 50 days and even after treatment with another protease (chymotrypsin) for 32 h, the performance of enolase digestion was successfully maintained. The use of EC-TR for the enolase digestion in the ultra-sonication system resulted in fast (∼10 min) and efficient digestions with reproducible performance under recycled uses.
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