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 inte...