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.