Effective Methods to Globally Analyze Cell Surface Proteins and Glycoproteins

摘要： 细胞表面蛋白和糖蛋白是哺乳动物细胞生存所必需的，它们经常调节 并代表细胞的不同发育和患病状态。许多重要的表面蛋白，如 作为受体、转运体和酶，作为治疗靶点具有特殊的临床意义，它们 也可以作为疾病检测的有价值的生物标志物。尽管表面蛋白非常重要 和糖蛋白，在它们的全球分析和有效的方法之间有很大的差距 实现它。这个项目的目标是通过设计来全球分析表面蛋白和糖蛋白 集点击化学、酶反应、质谱学于一体的创新有效方法-- 以蛋白质组学为基础。在强劲的初步数据指导下，这一目标将通过追求三个具体目标来实现 目标。(1)发展一种有效的表面蛋白质综合分析方法。细胞表面蛋白 活细胞将通过无铜点击化学有效地标记。细胞裂解后，表面蛋白与 特定的标签将通过第二个精心设计的点击反应共价结合到珠子上。共价键 连接将允许通过变性浓缩表面来完全去除非特定结合的蛋白质 蛋白质，用于特定和全球表面蛋白质鉴定。(2)鉴定细胞表面N-糖蛋白 全球范围内和特定地点的。将设计结合温和的酶反应和化学反应的新方法 靶向表面糖蛋白，包括含有特定糖蛋白的蛋白质，用于MS分析。(3)学习 并系统地定量测定癌细胞中的表面蛋白和糖蛋白。小说 这些方法将提供一个独特的机会来研究癌细胞的表面蛋白和糖蛋白。 渐进式侵袭。这项拟议的研究包含五个破译细胞表面的创新。 蛋白质组和糖蛋白质组。首先，细胞表面蛋白将通过无铜点击化学进行标记 反应为首次反应，反应速度快、专属性强、反应条件温和，是理想的反应条件。第二, 将使用共价相互作用将含有特定标签的表面蛋白质与磁性连接起来 珠子。这将使非特异结合蛋白能够完全去除，并克服长时间的 站立的障碍。第三，将系统地研究表面蛋白质动力学，并研究它们的半衰期。 量过了。第四，将实现对表面糖蛋白的全面和定点鉴定，以及 含有特定糖链的表面糖蛋白将被选择性地分析。第五，表面蛋白分泌 将在全球范围内进行研究，并对不同癌症的表面蛋白和糖蛋白进行量化 具有渐进侵袭性的细胞。这些方法将在生物医学研究中得到广泛应用 更好地了解表面蛋白的功能和疾病的分子机制，以及 协助发现表面蛋白作为药物靶点和有效的疾病检测生物标志物。

SUMMARY: Cell surface proteins and glycoproteins are essential for mammalian cell survival, and they frequently modulate and represent different developmental and diseased statuses of cells. Many important surface proteins, such as receptors, transporters and enzymes, are of extraordinary clinical interest as therapeutic targets, and they can also serve as valuable biomarkers for disease detection. Despite the critical importance of surface proteins and glycoproteins, there is a substantial gap between their global analysis and effective methods available to achieve it. The objective of this project is to globally analyze surface proteins and glycoproteins by designing innovative and effective methods integrating click chemistry, enzymatic reactions and mass spectrometry- based proteomics. Guided by strong preliminary data, this objective will be fulfilled by pursuing three specific aims. (1) Develop an effective method for the comprehensive analysis of surface proteins. Surface proteins in living cells will be efficiently tagged via copper-free click chemistry. After cell lysis, surface proteins with the specific tag will be covalently bound to beads via a second well-designed click reaction. The covalent connections will allow the complete removal of non-specifically bound proteins by denaturing enriched surface proteins, for specific and global surface protein identification. (2) Identify N-glycoproteins on the cell surface globally and site-specifically. New methods integrating mild enzymatic and chemical reactions will be designed to target surface glycoproteins, including proteins bearing a particular glycan, for MS analysis. (3) Study surface protein secretion and systematically quantify surface proteins and glycoproteins in cancer cells. Novel methods will provide a unique opportunity to investigate surface proteins and glycoproteins in cancer cells with progressive invasiveness. The proposed research contains five innovations to decode the cell surface proteome and glycoproteome. First, cell surface proteins will be tagged through a copper-free click chemistry reaction for the first time, which is ideal due to its high speed, specificity and mild reaction conditions. Second, covalent interactions will be employed to connect surface proteins containing the specific tag with magnetic beads. This will enable the complete removal of non-specifically bound proteins, and overcome the long- standing obstacle. Third, surface protein dynamics will be systematically studied and their half-lives will be measured. Fourth, comprehensive and site-specific identification of surface glycoproteins will be achieved, and surface glycoproteins containing a particular glycan will be selectively analyzed. Fifth, surface protein secretion will be globally investigated, and surface proteins and glycoproteins will be quantified among different cancer cells with progressive invasiveness. These methods will have extensive applications in biomedical research by providing a better understanding of surface protein functions and the molecular mechanisms of disease, and assisting in the discovery of surface proteins as drug targets and effective biomarkers for disease detection.