Robust trypsin coating on electrospun polymer nanofibers in rigorous conditions and its uses for protein digestion.

An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 and 50°C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 1.42 and 231 h at 40°C, respectively. The improved stability of EC-TR can be explained by covalent linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40°C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of 1 week. In the same condition, CA-TR showed poor performance from the beginning and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.

Trypsin coatings on electrospun and alcohol-dispersed polymer nanofibers for a trypsin digestion column.

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.