A mechanism-based ICAT strategy for comparing relative expression and activity levels of glycosidases in biological systems.
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.
Hekmat
Omid
O
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada.
He
Shouming
S
Warren
R Antony J
RA
Withers
Stephen G
SG
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018533:Biomass; D044602:Cellulases / Q000737:chemistry / Q000378:metabolism; D040143:Cellulomonas / Q000201:enzymology*; D003903:Deuterium; D004187:Disaccharides / Q000138:chemical synthesis / Q000737:chemistry; D005960:Glucosides / Q000138:chemical synthesis / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006859:Hydrogen; D007202:Indicators and Reagents; D007553:Isotope Labeling; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D012988:Soil Microbiology; D021241:Spectrometry, Mass, Electrospray Ionization; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004187:Disaccharides; D005960:Glucosides; D007202:Indicators and Reagents; D020543:Proteome; D006859:Hydrogen; D003903:Deuterium; D044602:Cellulases; D006026:Glycoside Hydrolases; D014995:Xylosidases