Synthesis and testing of mechanism-based protein-profiling probes for retaining endo-glycosidases

New functional proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Glycosidases play critical roles in biology, yet a robust method for functional analysis of their activities and identities in biological proteomes is still lacking. An aryl 2-deoxy-2-fluoro xylobioside inactivator was conjugated through cleavable and noncleavable linker arms to a biotin tag, thereby yielding two new active-site-directed reagents for activity-based profiling of retaining beta-glycanases in complex proteomes. Crucially, these tagged reagents possess high specificity for their target enzymes with kinetic parameters similar to those of the untagged reagent. Western blotting showed that these reagents bind and covalently label active retaining beta-glycanases both in pure enzyme samples and in the secreted proteome of the soil bacterium Cellulomonas fimi. Such reagents therefore show great promise for future activity-based targeting of glycanases.