Spatiotemporally Resolved Profiling of Protein Movement by TransitID.

Protein functions are tightly associated with their locations and can be dynamically regulated by movement between cellular regions. Traditional methods, such as fluorescent protein-based imaging and fractionation-coupled proteomics, have fundamental limitations in the unbiased discovery of protein trafficking events. Proximity labeling (PL) technology has emerged as a versatile tool in spatial proteomics based on genetically encoded promiscuous enzymes, which generate reactive species to tag endogenous proteins. Although it has been widely applied to provide snapshots of subcellular proteomes, traditional PL fails to effectively map dynamic protein trafficking. To overcome this challenge, TransitID was developed to map endogenous proteome trafficking with nanometer spatial resolution within and between living cells. Two orthogonal PL enzymes, TurboID and APEX2, are targeted to source and destination regions, respectively, followed by tandem PL to record protein movement. Here, we summarize the materials and experimental procedures for TransitID, enabling the analysis of protein trafficking between organelles and neighboring cells.