µMap Photoproximity Labeling on the Cell Surface

Understanding a protein's interactome provides crucial insight into its function and its contribution to disease. Traditional methods such as co‐immunoprecipitations often fail to capture interactions that are dependent on native cellular architecture such as those found on the cellular membrane. While enzyme‐based proximity labeling utilizing peroxidases or biotin ligases can achieve in situ interactome mapping, these approaches are limited by labeling radii, cellular engineering, and amino acid labeling biases. Antibody‐guided µMap photoproximity labeling addresses the constraints of these alternative platforms. Here, we apply µMap photoproximity labeling to study the interactome of HER2 by using antibody‐guided proximity labeling to target the endogenous protein, ablating the need for cellular engineering, and leveraging the advantages of µMap's short 4‐nm labeling radius. The protocols presented here describe the preparation of iridium‐antibody conjugates and its application in studying protein interactomes through mass‐spectrometry based analysis. While HER2 was used as a model in this article, this method is broadly applicable and can be used to study any cell surface protein with an appropriate commercially available antibody.