Abstract
Extracellular expression is essential for the function of secreted and cell surface proteins. Proper intracellular trafficking depends on protein interactions in multiple subcellular compartments. Co-immunoprecipitation and the yeast two-hybrid system are commonly used to investigate protein-protein interactions. These methods, however, depend on high-affinity protein interactions. In many glycoproteins, glycans are important for protein intracellular trafficking and extracellular expression. If glycoprotein interactions are transient and relatively weak, it may be challenging to use co-immunoprecipitation or the two-hybrid system to identify glycoprotein-binding partners. To circumvent this problem, protein cross-linking can be applied first to immobilize the transient and/or low-affinity protein interactions. Here we describe a protocol of protein cross-linking, co-immunoprecipitation, and proteomic analysis, which was used to identify endoplasmic reticulum (ER) chaperones critical for the folding and ER exiting of N-glycosylated serine proteases in human embryonic kidney (HEK) 293 cells. This approach can be used to identify other protein interactions in a variety of cells.