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

TitleSynthesis and testing of mechanism-based protein-profiling probes for retaining endo-glycosidases
Publication TypeJournal Article
Year of Publication2006
AuthorsWilliams, SJ, Hekmat, O, Withers, SG
JournalCHEMBIOCHEM
Volume7
Pagination116-124
Date PublishedJAN
ISSN1439-4227
Abstract

New functional proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Glycosidoses play critical roles in biology, yet a robust method for functional analysis of their activities and identities in biological proteomes is still locking. 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-glycanoses 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.

DOI10.1002/cbic.200500279