Multiprobe Photoproximity Labeling of the EGFR Interactome in Glioblastoma Using Red-Light

Photocatalytic proximity labeling has emerged as a valuable technique for studying interactions between biomolecules in a cellular context, providing precise spatiotemporal control over protein labeling. One significant advantage of these methods is their modularity, allowing the use of a single photocatalyst with different reactive probes to expand interactome coverage and capture diverse protein interactions. Despite these advances, fewer methods have been developed using red-light excitation, limiting the use of photoproximity labeling in more complex media such as tissues and animal models. Herein, we develop a platform for proximity labeling under red-light excitation, utilizing a single catalyst and two distinct probe types. We first design a carbene based labeling system that utilizes sulfonium diazo probes. This system is successfully applied on A549 cells to capture the interactome of epidermal growth factor receptor (EGFR) using a Cetuximab-Chlorin e6 conjugate. Benchmarking against established techniques indicates that this approach performs comparably to leading carbene-based proximity labeling methods. Next, we leverage the strong singlet oxygen generation (SOG) ability of Chlorin e6 to establish an alternative labeling system using aniline and hydrazide probes. EGFR directed chemoproteomics experiments reveal significant overlap with the carbene system, with the carbene approach capturing a subset of interactions identified by the SOG system. Finally, we deploy our approach for the characterization of EGFR in resected human glioblastoma (GBM) tissue samples removed from distinct locations in the same tumor, representing the tumor's infiltrating edge and its viable center, identifying several GBM specific interacting proteins that may serve as a launch point for future therapeutic campaigns.