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A mechanism-based ICAT strategy for comparing relative expression and activity levels of glycosidases in biological systems

TitleA mechanism-based ICAT strategy for comparing relative expression and activity levels of glycosidases in biological systems
Publication TypeJournal Article
Year of Publication2008
AuthorsHekmat, O, He, S, R. Warren, AJ, Withers, SG
JournalJOURNAL OF PROTEOME RESEARCH
Volume7
Pagination3282-3292
Date PublishedAUG
ISSN1535-3893
Abstract

An activity-based isotope-coded affinity tagging (AB-ICAT) strategy for proteome-wide quantitation of active retaining endoglycosidases has been developed. Two pairs of biotinylated, cleavable, AB-ICAT reagents (light H-8 and heavy D-8) have been synthesized, one incorporating a recognition element for cellulases and the other incorporating a recognition element for xylanases. The accuracy of the AB-ICAT methodology in quantifying relative glycosidase expression/activity levels in any two samples of interest has been verified using several pairs of model enzyme mixtures where one or more enzyme amounts and/or activities were varied. The methodology has been applied to the biomass-degrading secretomes of the soil bacterium, Cellulomonas fimi, under induction by different polyglycan growth substrates to obtain a quantitative profile of the relative expression/activity levels of individual active retaining endoglycanases per C. fimi cell. Such biological profiles are valuable in understanding the strategies employed by biomass-degrading organisms in exploiting environments containing different biomass polysaccharides. This is the first report on the application of an activity-based ICAT method to a biological system.

DOI10.1021/pr7008302