Cell Surface Engineering Enables Surfaceome Profiling.

ABSTRACT: Cell surface proteins (CSPs) are vital molecular mediators for cells and their extracellular environment. Thus, understanding which CSPs are displayed on cells, especially in different cell states, remains an important endeavor in cell biology. Here, we describe the integration of cell surface engineering with radical-mediated protein biotinylation to profile CSPs. This method relies on the prefunctionalization of cells with cholesterol lipid groups, followed by sortase-catalyzed conjugation with an APEX2 ascorbate peroxidase enzyme. In the presence of biotin-phenol and H₂O₂, APEX2 catalyzes the formation of highly reactive biotinyl radicals that covalently tag electron-rich residues within CSPs for subsequent streptavidin-based enrichment and analysis by quantitative mass spectrometry. While APEX2 is traditionally used to capture proximity-based interactomes, we envisioned using it in a "baitless" manner on cell surfaces to capture CSPs. We evaluate this strategy in light of another CSP labeling method that relies on the presence of cell surface sialic acid. Using the APEX2 strategy, we describe the CSPs found in three mammalian cell lines and compare CSPs in adherent versus three-dimensional pancreatic adenocarcinoma cells.

SUBMITTER: Vilen Z 

PROVIDER: S-EPMC9901301 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature