Effective Methods to Globally Analyze Cell Surface Proteins and Glycoproteins

总结： 细胞表面蛋白和糖蛋白是哺乳动物细胞生存所必需的，并且它们经常调节 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、转运蛋白和酶，作为治疗靶点具有非凡的临床意义，并且它们 也可以作为疾病检测的有价值的生物标志物。尽管表面蛋白至关重要 和糖蛋白，它们的全球分析和有效的方法之间存在很大的差距， 该项目的目标是通过设计一种新的表面蛋白质和糖蛋白的分析方法， 创新和有效的方法集成点击化学，酶促反应和质谱- 基于蛋白质组学在强有力的初步数据的指导下，将通过以下三个具体目标来实现这一目标： 目标。(1)建立一种有效的表面蛋白质综合分析方法。表面蛋白 活细胞将通过无铜点击化学被有效地标记。在细胞裂解后，表面蛋白与 特异性标记将通过第二个精心设计点击反应共价结合到珠上。共价 连接将允许通过使富集的表面变性而完全去除非特异性结合的蛋白质 蛋白质，用于特异性和全局表面蛋白质鉴定。(2)识别细胞表面的N-糖蛋白 全球和特定地点。将设计新的方法整合温和的酶和化学反应 靶向表面糖蛋白，包括带有特定聚糖的蛋白质，用于MS分析。(3)研究 表面蛋白分泌和系统定量癌细胞中的表面蛋白和糖蛋白。小说 方法将提供一个独特的机会，研究表面蛋白和糖蛋白的癌细胞， 渐进式入侵拟议的研究包含五项创新，以解码细胞表面 蛋白质组和糖蛋白质组。首先，细胞表面蛋白质将通过无铜点击化学标记 该方法具有快速、特异、反应条件温和等优点。第二、 将采用共价相互作用将含有特异性标记的表面蛋白与磁性标记连接。 珠这将能够完全去除非特异性结合的蛋白质，并克服长期的- 障碍。第三，将系统地研究表面蛋白质动力学， 测定了第四，将实现表面糖蛋白的全面和位点特异性鉴定， 将选择性地分析含有特定聚糖的表面糖蛋白。五、表面蛋白分泌 将在全球范围内进行研究，并将在不同的癌症中定量表面蛋白和糖蛋白 具有渐进性侵袭性的细胞。这些方法将在生物医学研究中有广泛的应用， 提供对表面蛋白功能和疾病分子机制的更好理解， 有助于发现表面蛋白作为药物靶标和用于疾病检测的有效生物标志物。

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.