(260e) Efficient Protein Digestion Using Highly-Stable Enzyme Coatings on Magnetic Nanofibers

Trypsin (TR), commonly used for protein digestion in proteomic analysis, was immobilized and stabilized on polymer nanofibers with superparamagnetic nanoparticles (magnetic nanofibers, NP-NFs). By electrospinning the homogeneous mixture of superparamagnetic nanoparticles (NPs) and polystyrene-poly(styrene-co-maleic anhydride), NPs were effectively entrapped within polymer nanofibers, generating magnetically-separable nanofibers with high surface area as an effective carrier for enzyme immobilization. Trypsin coatings on magnetic nanofibers (EC-TR/NP-NFs, and also called EC-TR), fabricated via simple crosslinking of TR molecules onto NP-NFs, were highly stable and could be easily recycled via facile magnetic separation of NP-NFs. EC-TR showed negligible loss of trypsin activity even after incubation in an aqueous buffer under rigorous shaking for 80 days, while the control samples of covalently-attached TR on NP-NFs (CA-TR/NP-NFs) and free TR lost more than 90 % of their initial activities within 11 and 6 days, respectively. 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 chymotrypsin for 32 hours, and 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 minutes) and efficient digestions with reproducible performance under recycled uses.