Characterization and enrichment of glycoprotein/glycan via multi-functional free radical reagents

项目摘要 糖蛋白是细胞外基质和细胞表面的普遍存在的组分， 据报道，在细胞-细胞和细胞-基质相互作用中具有独特的结构和功能作用。 据估计，超过50%的人类蛋白质是潜在糖基化的。聚糖的改变 糖蛋白的表达与许多疾病如癌症，糖尿病， 和免疫紊乱。同时，糖基化位点的鉴定也是至关重要的， 解读生理相关性和潜在诊断价值。因此表征 疾病相关的糖蛋白对于理解它们的功能至关重要， 分子水平上，从而有利于诊断性糖蛋白和/或聚糖的鉴定 生物标志物和更好的治疗药物设计。在过去的十年里，质谱 已成为聚糖和糖蛋白表征的关键技术。然而，（1） 聚糖部分的结构复杂性，2）难以确定糖基化位点，以及3）低- 大量的天然糖蛋白使得糖蛋白的质谱表征 特别具有挑战性。因此，在质量控制之前，糖蛋白和/或聚糖的有效富集是有利的。 糖蛋白的质谱分析和面部质谱分析是期望的， 糖蛋白的综合表征。注意到自由基在 以系统和可预测的方式解构聚糖和糖蛋白（初步数据）， 本研究的目标是开发固载自由基探针（SSFRP）， 聚糖部分富集和表征，以及自由基活化的糖蛋白 用于糖蛋白表征的结构解析（FRAGPSE）试剂，特别是 蛋白质测序和糖基化测定。拟议的研究活动将 一种同时富集和表征糖蛋白的简便工具，包括1） 从复杂混合物中富集糖蛋白和聚糖，2）分析蛋白质序列，3） 聚糖部分结构的阐明，和4）糖基化位点的鉴定。拟议 研究包括（1）SSFRP和FRAGPSE的设计和合成，（2）检查 SSFRP阐明聚糖结构的能力，（3）检查SSFRP 富集和表征从糖蛋白释放的聚糖，和（4）检查能力 以阐明糖蛋白的结构。总的来说， 所提出的方法将导致痕量糖蛋白的高通量表征， 允许糖蛋白的表征对于最终用户来说是广泛可及的和成本有效的。 这项研究将大大推动聚糖研究的前沿。

Project Summary Glycoproteins are ubiquitous components of extracellular matrices and cellular surfaces, and have been reported to have unique structural and functional roles in cell-cell and cell-matrix interactions. Over 50% of human proteins are estimated to be potentially glycosylated. Alterations of glycan expression in glycoprotein have been associated with many diseases, such as cancers, diabetes, and immune disorders. Meanwhile, the identification of glycosylation site is also crucial for deciphering physiological relevance and potential value as a diagnostic. Therefore characterization the disease-associated glycoproteins is essential for the understanding of their functions at a molecular level, and thus benefits the identification of diagnostic glycoprotein and/or glycan biomarkers and the better design of therapeutic drugs. Over the past decade, mass spectrometry has become the pivotal technique for glycan and glycoprotein characterization. However, 1) the structural complexity of glycan moiety, 2) difficulty in determining glycosylation sites, and 3) the low- abundance of native glycoproteins render the mass spectrometric characterization of glycoprotein especially challenging. Therefore, efficient enrichment of glycoprotein and/or glycans prior to mass spectrometry analysis, and facial mass spectrometric analysis of glycoprotein are desirable for the comprehensive characterization of glycoproteins. Noting that free radicals excel in the deconstruction of glycan and glycoprotein in a systematic and predictable fashion (preliminary data), the goal of this research is to develop solid-supported free radical probes (SSFRP) for glycan moiety enrichment and characterization, and free radial activated glycoprotein structure elucidation (FRAGPSE) reagent for glycoprotein characterization, especially the protein sequencing and glycosylation determination. The proposed research activities will bring an easily accessible tool to simultaneously enrich and characterize glycoproteins, including 1) enrichment of glycoprotein and glycans from complex mixtures, 2) analysis of protein sequences, 3) elucidation of glycan moiety structure, and 4) identification of the glycosylation sites. The proposed research includes (1) design and synthesis of SSFRP and FRAGPSE, (2) examination of the capability of SSFRP to elucidate glycan structure, (3) examination of the capability of the SSFRP to enrich and characterize glycans released from glycoproteins, and (4) examination of the capability of the FRAGPSE to elucidate the structures of glycoproteins. Overall, the successful completion of proposed approach will result in high-throughput characterization of trace amount of glycoproteins, allow the characterization of glycoproteins broadly accessible and cost-effective for the end-user. The proposed research will move the frontier of glycan study forward significantly.