A proximity-based protein labeling technology for high-resolution mapping of the HER2 interactome in breast cancer

Background

The study of protein-protein interactions, particularly in the context of membrane receptors like HER2 in breast cancer, is a critical area of biomedical research. HER2 (human epidermal growth factor receptor 2) is a receptor tyrosine kinase that is amplified in approximately 15–20% of breast cancers, where it drives tumor progression and is the target of several approved therapies. Despite the availability of targeted drugs, resistance to these therapies remains a significant clinical challenge, and the precise role of HER2 in cancers classified as HER2-negative is not fully understood. Mapping the HER2 interactome—the network of proteins that associate with HER2—can provide crucial insights into the mechanisms of drug resistance, cancer progression, and potential new therapeutic targets. However, the dynamic and spatially restricted nature of protein interactions at the cell membrane, as well as the transient nature of many signaling complexes, makes comprehensive interactome mapping technically challenging.

Traditional approaches to studying protein-protein interactions, such as immunoprecipitation (IP) and affinity purification-mass spectrometry (AP-MS), have notable limitations when applied to membrane proteins like HER2. These methods often require cell lysis and harsh extraction conditions, which can disrupt weak or transient interactions and fail to capture the true in situ microenvironment of the target protein. Additionally, membrane proteins are notoriously difficult to solubilize and purify without loss of interacting partners, leading to incomplete or biased interactome maps. Current proximity labeling techniques, while promising, can suffer from issues such as poor spatial resolution, limited control over labeling radius, and challenges in targeting labeling reagents specifically to the protein of interest. As a result, there is a pressing need for improved technologies that can capture the native interactome of membrane proteins with high spatial precision and sensitivity, particularly in live-cell contexts relevant to disease biology.

Technology

This proximity-based labeling technology is designed for high-resolution mapping of the HER2 interactome in human breast cancer cells. It utilizes conjugates that combine a transition metal catalyst, such as an iridium complex, with an anti-HER2 targeting agent like an antibody or kinase inhibitor. The system is engineered for tunable cell permeability and spatial targeting by modifying ligand substituents on the catalyst. Upon activation by visible light, the catalyst transfers energy to a protein labeling agent (such as diazirine or azide), generating a reactive intermediate (carbene or nitrene) that covalently labels proteins within a precisely defined nanometer-scale radius around HER2. These labeled proteins are functionalized with detectable markers (e.g., biotin), enabling their enrichment and identification through mass spectrometry. The platform, known as µMap, allows for quantitative and high-throughput profiling of protein-protein interactions, capturing both stable and transient associations in their native cellular context.

What differentiates this technology is its ability to overcome the limitations of traditional immunoprecipitation methods, which often struggle with membrane proteins and fleeting or spatially restricted interactions. By leveraging light-activated, proximity-based labeling, the system precisely maps the local protein environment of HER2, providing unprecedented spatial and temporal resolution. The tunable nature of the catalyst and labeling agents allows researchers to control the diffusion radius and cell permeability, adapting the system to various experimental needs. The platform’s validation across multiple breast cancer subtypes revealed not only established HER2 interactors but also novel proteins implicated in drug resistance, such as galectins and PTPRF, and demonstrated that targeting these interactors can restore drug sensitivity. This comprehensive, modular approach enables robust, reproducible interactome mapping and is broadly applicable to other receptor systems, making it a transformative tool for precision oncology and therapeutic discovery.

Benefits

• Enables high-resolution, proximity-based mapping of the HER2 protein interactome in live cells
• Captures transient and spatially restricted protein interactions that traditional methods miss
• Tunable catalyst and reactive intermediate properties allow precise control of labeling radius and cell permeability
• Facilitates identification of both known and novel HER2 interactors across diverse breast cancer subtypes
• Supports quantitative proteomic analysis through enrichment of labeled proteins with detectable markers
• Reveals mechanisms of drug resistance and identifies therapeutic targets, such as galectins and PTPRF
• Robust, reproducible workflow compatible with various labeling agents and downstream detection methods
• Broad applicability to other receptor systems and disease contexts, advancing precision oncology research

Applications

• HER2 interactome mapping in cancer
• Drug resistance mechanism discovery
• Precision oncology target identification
• High-throughput protein-protein interaction profiling

Additional info

This proximity-based labeling system utilizes conjugates of transition metal catalysts, such as iridium complexes, covalently linked to anti-HER2 targeting moieties. Upon visible-light activation, the catalyst transfers energy to protein labeling agents, generating reactive carbene or nitrene intermediates. These intermediates covalently modify proteins within a tunable nanometer-scale radius. Modular ligand substituents control cell permeability. Labeled proteins are enriched for mass spectrometry, enabling high-resolution mapping of the HER2 interactome.