Journal
JOURNAL OF PROTEOME RESEARCH
Volume 7, Issue 8, Pages 3282-3292Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr7008302
Keywords
activity-based ICAT; comparative proteomics; glycosidases; biomass conversion; mass spectrometry
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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.
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