Reagents to Investigate Glycosylated Macromolecules

研究糖基化大分子的试剂

• 批准号: 7216413
• 负责人: Doug Chan
• 金额: $ 30.09万
• 依托单位: PROTECH LABORATORIES, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-03-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7216413
• 关键词: Acetylene; Affinity; Amino Acid Sequence Databases; Azides; Biological; Biotin; Buffers; Cataloging; Catalogs; Cells; Condition; Detection; Development; Disease; Doctor of Philosophy; Evaluation; Family; Generations; Glass; Glucosamine; Glycosides; In Situ; In Vitro; Investigation; Link; Mass Spectrum Analysis; Methodology; Modification; Penetration; Phase; Phosphines; Physiological; Post-Translational Protein Processing; Preparation; Production; Property; Proteins; Proteomics; Radiolabeled; Reaction; Reagent; Research; Research Personnel; Role; Sepharose; Solid; System; Technology; Variant; Western Blotting; amidation; analog; base; chemical synthesis; covalent bond; improved; macromolecule; nano; novel; phosphine; programs; radiotracer; tissue culture; tool; triphenylphosphine

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.

描述（由申请人提供）：本II期申请的总体目标是扩展和优化TAGTM（通过叠氮基糖苷标记）技术，用于选择性检测、定量、衍生化和/或分离新O-GlcNAc修饰的蛋白质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，并将加快新的药理学靶点的鉴定。TAGTM技术由三个步骤组成：（1）化学合成细胞可渗透的GlcNAc叠氮化物，并将其掺入细胞中，而不是GlcNAc（蛋白质O-GlcNAc修饰的天然底物）。(2)GlcNAc叠氮化物修饰的蛋白质与膦捕获试剂的选择性原位缀合产生稳定的共价键。缀合或标记反应利用了O-GlcNAc叠氮化物修饰的蛋白质上的叠氮基与捕获试剂的膦之间的良好先例和容易的施陶丁格反应，随后是分子内酰胺化。和（3）缀合产物的检测、定量和/或分离。膦捕获试剂可以连接到，阿利亚是，（i）生物素或其他亲和试剂;（ii）固体支持物（例如，玻璃或琼脂糖珠），具有或不具有可光裂解的接头;和（iii）荧光的、放射性标记的或其它分析化合物。在初步研究和I期研究期间，我们（a）完成了全乙酰化N-（2-叠氮乙酰基）葡糖胺（全乙酰化GlcNAc叠氮化物）和膦捕获试剂的化学合成;（B）制备了三种化学上不同的生物素化捕获试剂，即，三苯基膦、硫代膦酯和末端炔属捕获试剂;（c）在体外实现了合成的GlcNAc叠氮化物和生物素化的三苯基膦捕获试剂之间的高产率缀合反应;（d）证明了利用组织培养中的细胞将全乙酰化的GlcNAc叠氮化物掺入到蛋白质中;（e）比较了三种类型的捕获试剂用于缀合至O-GlcNAc叠氮化物修饰的蛋白质的功效;和（f）利用TAGTM通过nano-HPLC/LCQ质谱和蛋白质序列数据库比较从CHO-K1细胞中分离和鉴定110个O-GlcNAc叠氮化物修饰的蛋白质。在第二阶段，我们将显着扩展和完善我们的初步进展，包括改进化学合成的变体O-GlcNAc叠氮化物底物和膦捕获试剂具有更好的酶，物理和反应特性，以及验证这些工具的蛋白质组学和生物医学意义的分析问题。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修饰的蛋白质。