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.