Digitonin-Based Permeabilization Enables Rapid Enrichment of Membrane Proteins for Deep Proteomics

Abstract Membrane proteins (MPs) govern molecular transport, signal transduction, and intercellular communication and are central to mechanistic biology, biomarker discovery, and therapeutic targeting. Their hydrophobicity, low abundance, and poor solubility, however, continue to complicate efficient recovery and deep proteomic analysis. Here, we developed a digitonin-mediated permeabilization workflow for rapid MPs enrichment and systematically optimized digitonin concentration, incubation temperature, and treatment duration. Under the optimized condition (0.01% digitonin at 37 °C for 30 min), soluble cytosolic proteins were efficiently released while membrane-associated material was retained for subsequent detergent extraction. Across two independent THP-1 membrane-proteomics experiments, approximately 75−78% of proteins that were more abundant in the digitonin-enriched fraction than in total cell lysate were annotated as MPs. The workflow reduces sample handling and can be completed in approximately 2 h. Marker-based validation in HeLa cells, primary human PBMCs, and pathogen-infected THP-1 macrophages further supported effective cytosolic release and membrane retention in additional cellular contexts. Together, these results establish digitonin permeabilization as a simple and practical front-end strategy for membrane proteomics, while its performance in additional primary and disease-derived specimens remains to be defined.