Development and Deployment of an Activity-Based N-Glycan Hydrolase Probe Suitable for Droplet-Based Screening

N-Glycans play essential roles in diverse biological processes, and enzymes capable of precisely modifying them offer powerful means to modulate their function for therapeutic applications. However, the discovery of such enzymes is hindered by the limited availability of sensitive, fully representative substrates that accurately mimic native N-glycan structures. Here, we report a fluorescence-quenched (FQ) N-glycan probe, chemoenzymatically synthesized from egg-derived sialoglycopeptide (SGP), that addresses this limitation. The probe remains nonfluorescent ("OFF") until enzymatic cleavage of a linkage within the intact complex type N-glycan substrate, which in turn triggers a fluorescent signal ('ON'). Incubation of the FQ N-glycan probe with a panel of glycoside hydrolases (GHs) reported to target specific linkages within complex type N-glycans, results in an increase in fluorescence, which can be readily monitored. Furthermore, we show that this probe is compatible with single-emulsion droplet-based screening and confirm its utility through a trial screen that can recover N-glycan degrading clones from mixed microbial populations using fluorescence activated single-droplet dispensing (FASD). While demonstrated for egg-derived SGP, this chemoenzymatic strategy may be extended to diverse glycans and glycopeptide substrates, providing a strategy to generate fully representative glycan probes for ultrahigh-throughput screening to advance both glycoengineering and fundamental glycomics research.