Effective MS-Based Methods for Unraveling Cell Surface Protein Interactions

摘要 哺乳动物细胞表面含有许多重要的蛋白质，包括受体和转运蛋白。他们 调节许多细胞事件，如感知细胞外营养物质，并启动细胞信号。 通常，这些蛋白质不是独立工作，而是相互协作完成任务。 细胞表面异常的蛋白质相互作用会影响蛋白质的活性，导致人类疾病，如 就像癌症一样。因此，识别和量化表面蛋白是不够的。有系统地调查 表面蛋白质相互作用有助于深入了解蛋白质的功能和细胞活动，以及 提供对人类疾病的分子机制的见解。然而，这是非常具有挑战性的 仅在细胞表面全面分析蛋白质的相互作用。这个项目的目标是开发 创新和有效的方法来全局分析细胞表面的蛋白质相互作用，特别是直接 低丰度表面蛋白之间的相互作用，并应用于表面蛋白及其相互作用的研究 上皮向间充质转化(EMT)过程中相互作用的变化。在强劲的初步数据的指引下， 这一目标将通过追求三个具体目标来实现。1)有效的基于MS的全局分析方法 细胞表面的蛋白质相互作用。通过发展创新的方法，表面蛋白 将对交互作用进行系统的分析。具有化学手柄的可切割交联剂将允许 交联肽的选择性浓缩，这将使我们能够识别更多的直接相互作用 在低丰度表面蛋白中。此外，将可分解洗涤剂集成到工作流程中将 增加细胞表面膜蛋白的覆盖率。2)基于自由基化学的标记方法 用于研究它们之间相互作用的表面蛋白。基于细胞表面覆盖有多聚糖的事实， 自由基将通过多聚糖的酶催化氧化而产生，它们将被用来快速标记表面 蛋白质。基于自由基化学的方法将进一步增加表面蛋白及其 互动。更全面地分析表面蛋白质及其在细胞表面的相互作用将是 已实现。3)细胞表面糖蛋白的定量和表面蛋白相互作用重塑 急诊室。在EMT过程中，细胞的性质发生了巨大的变化，如细胞形态、细胞与细胞之间的相互作用、 以及细胞流动性，这通常是由表面蛋白决定的。建议的方法将应用于 综合分析细胞表面糖蛋白与细胞表面蛋白相互作用网络重构 急诊室。这一结果将揭示EMT及其相关疾病的分子机制。如果没有 样本限制条件下，所提出的方法可广泛应用于研究表面蛋白质相互作用， 从而将表面蛋白确定为疾病生物标记物和药物靶标。

SUMMARY The surface of mammalian cells contains many important proteins including receptors and transporters. They regulate numerous cellular events, such as sensing extracellular nutrients, and initiating cell signaling. Frequently, these proteins do not work independently, but cooperate with one another instead to fulfill the tasks. Aberrant protein interactions on the cell surface will impact protein activities and result in human diseases such as cancer. Therefore, it is not sufficient to identify and quantify surface proteins. Systematic investigation of surface protein interactions can aid in an in-depth understanding of protein functions and cellular activities, and provide insights into the molecular mechanisms of human diseases. However, it is extraordinarily challenging to comprehensively analyze protein interactions only on the cell surface. The objective of this project is to develop innovative and effective methods to globally analyze protein interactions on the cell surface, especially direct interactions among low-abundance surface proteins, and apply the methods to study surface proteins and their interaction changes during the epithelial-to-mesenchymal transition (EMT). Guided by strong preliminary data, this objective will be fulfilled by pursuing three specific aims. 1) Effective MS-based methods for global analysis of protein interactions on the cell surface. Through the development of the innovative methods, surface protein interactors will be systematically analyzed. The cleavable cross-linker with a chemical handle will allow for selective enrichment of cross-linked peptides, which will enable us to identify many more direct interactions among low-abundance surface proteins. Furthermore, integrating cleavable detergents into the workflow will increase the coverage of membrane proteins on the cell surface. 2) Radical chemistry-based methods to tag surface proteins for studying their interactions. Based on the fact that the cell surface is covered with glycans, radicals will be generated through the enzymatic oxidation of glycans, and they will be used to quickly tag surface proteins. Radical chemistry-based methods will further increase the coverage of surface proteins and their interactions. More comprehensive analysis of surface proteins and their interactions on the cell surface will be achieved. 3) Quantification of surface glycoproteins and the surface protein interaction remodeling during the EMT. During the EMT, the properties of cells change dramatically, such as cell morphology, cell-cell interactions, and cell mobility, which are often determined by surface proteins. The proposed methods will be applied to comprehensively analyze surface glycoproteins and the surface protein interaction network remodeling during the EMT. The results will unveil the molecular mechanisms of the EMT and the EMT-relevant diseases. Without sample restrictions, the proposed methods can be extensively applied to study surface protein interactions, leading to the identification of surface proteins as disease biomarkers and drug targets.