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The construction of a trypsin column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coatings, which were prepared by a two-step process of covalent attachment and enzyme cross-linking. In a comparative study with the trypsin coatings on as-spun and nondispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than 20 min. By applying the alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the trypsin coatings were improved, yielding more complete and reproducible digestions. Regardless of alcohol dispersion or not, trypsin coatings showed better digestion performance and improved performance stability under recycled uses than covalently attached trypsin, in-solution digestion, and commercial trypsin beads. The combination of highly stable trypsin coatings and alcohol dispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for online and automated protein digestion.

Trypsin Coatings on Electrospun and Alcohol-Dispersed Polymer Nanofibers for a Trypsin Digestion Column