TaewonKo (Center for ProteoGenome Research, Department of Chemistry, Korea University, Seoul 02841)

TaewonKo (Center for ProteoGenome Research, Department of Chemistry, Korea University, Seoul 02841)

Cell-surface proteins play essential roles in numerous physiological processes, including signal transduction, ion transport, and intercellular communication. Due to their membrane localization and functional relevance, they have emerged as prominent targets in drug discovery and therapeutic development. This underscores the need for advanced proximity labeling strategies capable of selectively and accurately profiling membrane-bound proteins.

In this study, we developed a novel proximity labeling approach utilizing Desthiobiotin-phenol (DBP) in combination with wheat germ agglutinin–horseradish peroxidase (WGA-HRP) coating. This strategy enabled selective biotinylation of cell-surface proteins and facilitated the identification of previously uncharacterized biomarkers associated with metastatic processes. To rigorously assess the specificity of surface labeling, we performed comparative analyses of proteins labeled by endogenous peroxidase activity under both basal and nutrient-deprived conditions, serving as critical controls for background signal.

Biotinylated and DBP-labeled peptides were enriched via streptavidin-based immunoprecipitation and subsequently analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS), employing our proprietary dual online ultrahigh-pressure liquid chromatography (DO-UHPLC) with a 3-hour gradient. Manual inspection of peptide-spectrum matches (PSMs) ensured high-confidence identification of DBP-modified peptides. Importantly, peptides attributed to endogenous peroxidase activity were reliably identified and excluded from downstream analysis, thereby refining the surface proteome and enhancing the specificity and accuracy of cell-surface protein annotation.