Proteases, enzymes that catalyze the hydrolysis of amide bonds in peptides and proteins, are ubiquitous across all forms of life and affect every protein in its natural life cycle. Engineering programmed specificity onto stable protease scaffolds holds promise as a route to a new generation of useful enzymes with requisite biotechnological properties. One of the long-standing applications of proteases has been in generating peptides for mass spectrometry (MS) analyses in proteomic experiments. Since post-translational modifications (PTMs) can be labile, proteomic techniques relying on tandem MS have difficulty in achieving comprehensive coverage of PTM sites within proteins. Proteases which selectively recognize PTMs may improve their identification in complex proteome samples and are easily adaptable to current proteomic methodologies. In the current work, we have utilized a combinatorial approach to engineer the substrate binding pocket of chymotrypsin, and isolate variants that can identify Asparagine (Asn)-linked glycosylation sites. With the aid of our display methodology, we screened large libraries (&gt;10<sup>7 </sup>variants) of chymotrypsin for specific proteolytic cleavage after Asn residues. In comparison to wild-type chymotrypsin, the engineered variant, NV3, displayed a 2 x 10<sup>4</sup>-fold reversal in catalytic selectivity for Asn at P1 position over tyrosine.  The applicability of NV3 in proteomic studies and identification of Asn-linked glycosylation sites was validated in MS analyses of full length proteins, RNAse A and Invertase.   Further, the specificity of NV3 to asparagine residues allowed for mapping of glycan sites of HEK293 proteins with improved sequence coverage. Our surface-display assisted combinatorial engineering methodology can readily be extended to modifying specificity of proteases and simplify identification and characterization of a broad range of PTMs.

Engineering substrate specificity of chymotrypsin for mass spectrometry based proteomics

251st National Meeting (2016)

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