Reagents to Investigate Glycosylated Macromolecules

描述（由申请人提供）：该II期申请的总体目标是扩展和优化TAGTM（通过叠氮糖苷标记）技术，用于选择性检测、定量、衍生化和/或分离新的O-GlcNAc修饰蛋白及其在蛋白质组学研究中的应用。这种强大的方法将加快我们对O-GlcNAc蛋白修饰的功能作用和生理意义的理解，并将加快识别新的药理靶点。TAGTM技术包括三个步骤：(1)化学合成细胞可渗透的GlcNAc叠氮化物，并将其掺入细胞中，代替O-GlcNAc修饰蛋白质的天然底物GlcNAc。(2) GlcNAc叠氮化物修饰的蛋白质与膦捕获试剂选择性原位偶联，形成稳定的共价键。偶联或标记反应利用了O-GlcNAc叠氮化物修饰蛋白上的叠氮化物基团与捕获试剂的磷化氢之间的预先和容易的Staudinger反应，然后进行分子内酰胺化。(3)共轭产物的检测、定量和/或分离。磷化氢捕获试剂可连接到(i)生物素或其他亲和试剂；（ii）固体支撑（如玻璃或蔗糖珠），含或不含可光解连接剂；（iii）荧光、放射性标记或其他分析化合物。在初步研究和I期研究中，我们(a)完成了过乙酰化N-（2-叠氮乙酰化）氨基葡萄糖（过乙酰化GlcNAc叠氮化物）和磷化氢捕获试剂的化学合成；(b)制备了三种化学性质不同的生物素化捕获试剂，即三苯基膦、膦硫酯和末端乙炔捕获试剂；(c)合成的GlcNAc叠氮化物与生物素化的三苯基膦捕获试剂在体外实现了高收率的偶联反应；(d)证明了过乙酰化GlcNAc叠氮化物在组织培养细胞中与蛋白质的结合；(e)比较三种捕获试剂偶联O-GlcNAc叠氮化物修饰蛋白的效果；(f)利用TAGTM通过纳米hplc /LCQ质谱法和蛋白质序列数据库比对，从CHO-K1细胞中分离鉴定110个O-GlcNAc叠氮修饰蛋白。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叠氮化物底物变体的化学合成和具有更好的酶、物理和反应性特性的磷化氢捕获试剂，并验证这些工具用于具有生物医学意义的蛋白质组学和分析问题。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.