Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation

Abstract

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.

Making the cut with protease engineering

“…Structure-guided focused mutagenesis libraries generally produce higher-fitness proteases compared to randomized variants. This especially applies to proteases with extensive substrate-binding sites such as trypsin (Tran et al, 2016), chymotrypsin (Ramesh et al, 2019), and BoNT (Blum et al, 2021). Indeed, the authors of this review also attempted large-scale screening efforts with the highly specific BoNT protease.…”

“…However, a carefully curated selection of mutations, with some residues identified by epPCR, more efficiently reshaped the enzyme's specificity from SNAP25 to SNAP23 (Dyer et al, 2020). Thus, random mutagenesis can identify nonobvious residues influencing protease specificity and catalysis (Tran et al, 2016;Packer et al, 2017;Ramesh et al, 2019), and is the standard approach to improving protease stability. Interestingly, the random mutagenesis strategies surveyed here are rarely followed up with focused mutagenesis.…”

“…Mapping glycan PTMs represents another important goal in proteomics. Varadarajan and coworkers engineered trypsin's homolog, chymotrypsin (S01.152), to recognize and cleave unmodified asparagine residues to reveal potential N-linked glycosylation sites (Ramesh et al, 2019). A FACS-based E coli display platform identified chymotrypsiN, which features eight added mutations and enables the identification of glycosylated asparagines for MS assays (Figure 4A).…”

“…Alternatively, evaluating orthologous protease sequences identifies residues potentially influencing catalysis or substrate specificity. For example, comparing chymotrypsin (S01.152) with related serine proteases with different selectivities revealed 11 residues that enabled morphing chymotrypsin into an asparagine-targeting protease through site-saturation mutagenesis (Ramesh et al, 2019). One useful tool, the HotSpot Wizard, mines bioinformatics data to suggest target sites for mutagenesis (Sumbalova et al, 2018).…”

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…Multiple protease approaches have already proven their benefit for glycoproteome characterization (86,(462)(463)(464)(465)(466). Even so, these largely rely on canonical proteases like trypsin, chymotrypsin, GluC, and AspN, among others, and some classes of glycosylation, e.g., mucintype O-glycosylation remain impervious to these proteases.…”