Protein-Corona-Based Nanomagnetic Enrichment Strategy for In-Depth Secretome Profiling of Lung Adenocarcinoma Organoids

Secreted proteins are critical mediators of intercellular
communication
that shape the tumor microenvironment (TME). While patient-derived
organoids (PDOs) provide physiologically relevant models that preserve
patient heterogeneity, comprehensive secretome profiling remains challenging.
This difficulty arises from the low abundance of secreted proteins
and high interference from exogenous media components essential for
organoid maintenance. Here, we report a Protein corona nanomagnetic
bead-based PDO Secretome Profiling workflow (PPSP) that integrates
magnetic-bead enrichment with high-resolution mass spectrometry data-independent
acquisition (HRMS–DIA). By leveraging the protein corona effect
to mitigate background interference, PPSP significantly enhances detection
depth. In a proof-of-concept application to lung adenocarcinoma (LUAD)
PDOs, the workflow identified 1376 proteins in a single samplea
4.9-fold increase over direct analysisand uncovered 1095 proteins
undetectable by conventional methods. Notably, PPSP enabled the detection
of both known LUAD biomarkers and previously uncharacterized secreted
proteins associated with patient survival. This robust and scalable
platform facilitates the in-depth profiling of the PDO secretome and
the identification of clinically relevant signaling proteins.