The overall objectives of this Phase II application are the extension and optimization of TAGTM (tagging via azido glycosides) technology for the selective detection, quantification, derivatization, and/or isolation of newly O-GlcNAc modified proteins and its application to proteomics studies. This powerful methodology will expedite our understanding of the functional role and physiological significance of O-GlcNAc protein modifications and will expedite the identification of novel pharmacological targets. TAGTM technology consists of three steps: (1) the chemical synthesis of cell permeable GlcNAc azides and their incorporation into cells instead of GlcNAc, the natural substrate for the O-GlcNAc modification of proteins. (2) Selective in situ conjugation of the GlcNAc azide-modified proteins with a phosphine capture reagent resulting in a stable covalent bond. The conjugation or tagging reaction exploits the well precedented and facile Staudinger reaction between the azide group on the O-GlcNAc azide-modified protein and the phosphine of the capture reagent followed by intramolecular amidation. And (3) the detection, quantification, and/or isolation of the conjugated products. The phosphine capture reagent can be linked to, inter alia, (i) biotin or other affinity reagents; (ii) solid supports (e.g., glass or sepharose beads) with or without a photocleavable linker; and (iii) fluorescent, radiolabeled, or other analytical compounds. In preliminary studies and during Phase I research, we (a) completed the chemical syntheses of peracetylated N-(2-azidoacetyl)giucosamine (peracetylated GlcNAc azide) and a phosphine capture reagent; (b) prepared three chemically different biotinylated capture reagents, viz., triphenylphosphine, phosphinothioester, and terminal acetylenic capture reagents; (c) achieved the high yield conjugation reaction in vitro between synthetic GlcNAc azide and the biotinylated triphenylphosphine capture reagent; (d) demonstrated the incorporation of peracetylated GlcNAc azide into proteins utilizing cells in tissue culture; (e) compared the efficacy of the three types of capture reagents for conjugation to O-GlcNAc azide-modified proteins; and (f) exploited TAGTM for the isolation and identification of 110 O-GlcNAc azide-modified proteins from CHO-K1 cells via nano-HPLC/LCQ mass spectrometry and protein sequence database comparisons. During Phase II, we shall significantly extend and refine our initial progress to include improved chemical syntheses of variants of O-GlcNAc azide substrates and phosphine capture reagents with better enzymatic, physical and reactivity properties as well as validate these tools for proteomic and analytical problems of biomedical significance. These studies will result in a family of reagents or kits for the characterization and quantification of O-GlcNAc modified proteins in various biological systems and diseases. ? ? ?
