The overall objectives of this application are the development of TAG(TM) (tagging via azido glycosides) reagents for the selective detection, quantification, derivatization, and/or isolation of O-GlcNAc-modified proteins and the validation of TAG(TM) reagents in proteomic studies. The overall process consists of three steps: (1) chemical synthesis of cell permeant GIcNAc azides and their enzymatic incorporation into proteins instead of the natural substrate, GIcNAc; (2) selective in situ conjugation of the GIcNAc azide-modified proteins with a capture reagent (phosphine or acetylene) resulting in a stable covalent bond; and (3) detection, quantification, and/or isolation of the conjugated products. In preliminary studies, we demonstrated the chemical syntheses of peracetylated N-(2-azidoacetyl) glucosamine (peracetylated GIcNAc-azide) and a phosphine capture reagent linked to biotin. We have also demonstrated that cellular proteins can be specifically labeled with peracetylated N-(2-azidoacetyl) glucosamine (peracetylated GIcNAc azide) and can be detected by streptavidin HRP following conjugation with a biotinylated phosphine capture reagent. The Phase I goals are: a) Chemical syntheses of improved phosphine and acetylenic capture reagents and comparisons of their conjugation efficacies. b) Evaluation of biotin-linked capture reagents for the detection of O-GIcNAc modified proteins. In Phase II, we will refine and extend our initial work to: (1) prepare variants of O-GIcNAc azide substrates with improved cellular penetration and incorporation into proteins; (2) optimize the selectivity, reaction kinetics, stability, and large scale synthesis of the capture reagent deemed most suitable in the comparisons during Phase I; (3) attach the optimized capture reagent to a solid matrix via a photocleavabie linker to expedite the isolation of O-GIcNAc-azide modified proteins; and (4) attach the optimized capture reagent to fluorescent, affinity, radiolabeled, or other analytical reagents for the detection, isolation, and/or quantification of O-GIcNAc-azide modified proteins.
