Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion

Abstract

A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in "real-world" proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

Figure 1

(a) Schematic diagram for the preparation of covalently-attached trypsin (CA-TR) and trypsin-aggregate coated nanofibers (EC-TR) on the PS:PSMA nanofibers. SEM images of (b) bare nanofibers, (c) CA-TR, and (d) EC-TR. (e) A zoomed-in SEM image of EC-TR.

Figure 2

Enzyme stability of free trypsin (empty triangles), covalently-attached trypsin (CA-TR, filled circles), and trypsin-aggregate coatings (EC-TR, empty circles) under repeated use and rigorous shaking (200 rpm). Inserted figure shows the enzyme stability during the first 12 days.

Figure 3

Comparison of the enzyme stability of covalently-attached trypsin (CA-TR, filled circles) and trypsin-aggregate coatings (EC-TR, empty circles) in the presence of α-chymotrypsin.

Figure 4

Time course analyses of different immobilization methods by iterative digestions of BSA across several days as a function of (a) protein sequence coverage; (b) number of identified BSA peptides using covalently-attached trypsin (CA-TR, filled circles) or trypsin-aggregate coatings (EC-TR, empty circles).

Figure 5

BSA digestion using covalently-attached trypsin (CA-TR) and trypsin-aggregate coatings (EC-TR) after 46 days of iterative use. (a) Comparison of CA-TR and EC-TR on the base peak chromatogram (trace of ions) corresponding to the charge +2 peptide LVVSTQTALA. (b) Mass spectrum showing the charge +1 and +2 of the peptide LVVSTQTALA from the BSA digestion using EC-TR. (c) MS/MS spectrum of +2 peptide LVVSTQTALA from the BSA digestion using EC-TR; asterisks mark the fragment peaks matching the theoretical ion masses from the y or b-series.

Figure 6

Application of trypsin-aggregate coated nanofibers and solution phase trypsin to the digestion of a whole yeast protein extract. (a) Score distributions from the search of all datasets against swissprot.fasta database (filled circles) or against the inverted database (empty circles). Curves were generated with an assumption of a Gaussian distribution. (b) Comparison of the number of identified peptides at a given FDR following the Spectrum Mill analysis. EC-TR nanofibers batch 1 (filled circles), EC-TR nanofibers batch 2 (empty circles), solution phase trypsin (empty triangles). (c) Representative chromatograms for the replicates of yeast proteome digestion using two different batches of EC-TR nanofibers. (d) Overlap of the identified proteins.