µMap Photoproximity Labeling on the Cell Surface
Year: 2025
Type: article
Abstract: 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. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and validation of iridium-antibody conjugates Basic Protocol 2: Proximity labeling and streptavidin enrichment for mass spectrometry.
Source: Current Protocols
DOI: 10.1002/cpz1.70216
Language: English
Topic: Biotin and Related Studies
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,890
OpenAlex ID: W4414952123