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 H 2 O 2 , 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 proximitybased 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.

Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling

“…PDPL was also utilized to characterize subcellular proteomes, where the specificity and proteome coverage were at least comparable with other proximity labeling methods and organelle-specific chemical probe-based methods. Proximity labeling has also been successfully applied in characterizing surfacome, lysosome proteome, and secretory pathway-associated proteomes 46,47 . We believe PDPL would be compatible with these subcellular organelles.…”

Surfaceome Profiling Identifies Basigin‐Chaperoned Protein Clients

“…We initially screened the WT LX‐2 cells for compatibility with the surfaceome profiling approach. In our previous work, we determined that optimization of biotin‐phenol incubation periods in each cell line was crucial in order to select CSPs over intracellular proteins [22] . Thus, we varied biotin‐phenol incubation times (1–5 min) during live cell labeling, and the resulting cell lysates were processed by ultracentrifugation to separate membrane‐bound and intracellular components.…”

“…In this method, an engineered ascorbate peroxidase enzyme (APEX2) is localized to the cell surface via a synthetic GGGYC peptide-modified cholesterol tether and an engineered sortase enzyme (eSrtA) [20] to facilitate the biotinylation of nearby electron-rich residues. [19] These biotin handles can be used to enrich the proteins for subsequent identification by quantitative mass spectrometry (MS)-based proteomics. Importantly, this method does not rely on the presence of specific post-translational modifications (PTMs) to reduce biases in the enrichment method.…”

“…We recently developed an approach to identify CSPs using cell surface engineering [18] and a “baitless” proximity tagging [19] strategy (Figure 1C). In this method, an engineered ascorbate peroxidase enzyme (APEX2) is localized to the cell surface via a synthetic GGGYC peptide‐modified cholesterol tether and an engineered sortase enzyme (eSrtA) [20] to facilitate the biotinylation of nearby electron‐rich residues [19] . These biotin handles can be used to enrich the proteins for subsequent identification by quantitative mass spectrometry (MS)‐based proteomics.…”

Veneer Is a Webtool for Rapid, Standardized, and Transparent Interpretation, Annotation, and Reporting of Mammalian Cell Surface N-Glycocapture Data

“…While a subset of cell surface N-glycocapture studies used trypsin only to liberate nonglycosylated peptides, that workflow does not provide experimental evidence of localization. 12 Of the 110 lysine capture methods, only six use the mass tag for evidence of localization. Overall, of the 469 total methods described across 424 publications, 77 were generated with approaches that use experimental evidence to assert surface localization of identified proteins, while 392 inferred subcellular localization based only on ontologies and annotations (Figure 1G).…”