Reagents to Investigate Glycosylated Macromolecules

描述(由申请人提供)：此第二阶段申请的总体目标是扩展和优化TAGTM(通过叠氮苷标记)技术，以选择性地检测、定量、衍生化和/或分离新的O-GlcNAc修饰的蛋白质及其在蛋白质组学研究中的应用。这一强大的方法学将加速我们对O-GlcNAc蛋白修饰的功能作用和生理意义的理解，并将加速识别新的药理靶点。TAGTM技术包括三个步骤：(1)化学合成细胞通透性的GlcNAc叠氮化合物，并将其掺入细胞内，而不是蛋白质O-GlcNAc修饰的天然底物GlcNAc。(2)将GlcNAc叠氮修饰的蛋白质与膦捕捉剂选择性原位偶联，形成稳定的共价键。偶联或标记反应利用了O-GlcNAc叠氮修饰蛋白上的叠氮基与捕捉剂的膦之间的简单的Staudinger反应，然后进行分子内酰胺化。和(3)共轭产物的检测、定量和/或分离。膦捕捉剂可以连接到(I)生物素或其他亲和剂；(Ii)具有或不具有可光裂解连接体的固体载体(例如，玻璃或琼脂糖珠)；以及(Iii)荧光、放射性标记或其他分析化合物。在初步研究和第一阶段研究中，我们(A)完成了过乙酰化N-(2-叠氮乙酰基)葡萄糖胺(过乙酰化GlcNAc叠氮)和膦捕捉剂的化学合成；(B)制备了三种化学上不同的生物素化捕捉剂，即三苯基膦、硫代膦和末端乙炔捕捉剂；(C)合成的GlcNaazide和生物素化的三苯基膦捕捉剂在体外实现了高产率的偶联反应；(D)在组织培养中证明了过乙酰化的Glcnacazide进入细胞；(E)比较了三种不同类型的捕捉剂对O-Glccazide修饰的蛋白质的偶联效果；和(F)开发了TAGTM，通过纳米高效液相色谱/LCQ质谱仪和蛋白质序列数据库比对，从CHO-K1细胞中分离和鉴定了110个O-GlcNAc叠氮化修饰的蛋白质。在第二阶段，我们将显著扩展和完善我们的初步进展，包括改进的O-GlcNAc叠氮化物底物和膦捕捉剂的化学合成，具有更好的酶性能、物理性能和反应性能，并验证这些工具用于具有生物医学意义的蛋白质组和分析问题。这些研究将产生一系列试剂或试剂盒，用于表征和定量各种生物系统和疾病中的O-GlcNAc修饰蛋白。

DESCRIPTION (provided by applicant): 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.