Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation

We have engineered the substrate specificity of chymotrypsin
to
cleave after Asn by high-throughput screening of large libraries created by comprehensive
remodeling of the substrate binding pocket. The engineered variant
(chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity
with an expanded preference for Asn-containing substrates. We confirmed
that protein engineering did not compromise the stability of the enzyme
by biophysical characterization. Comparison of wild-type ChyB and
ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative
and quantitative differences in the nature of the peptides and proteins
identified by liquid chromatography and tandem mass spectrometry.
ChyB-Asn enabled the identification of partially glycosylated Asn
sites within a model glycoprotein and in the extracellular proteome
of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit
of proteases to aid the mapping of N-linked glycosylation sites within
proteins and proteomes.