Spatial mapping of cell-surface protein glycosylation at molecular resolution

Cell-surface protein glycosylation is a fundamental post-translational modification that plays a crucial role in membrane protein function and cellular behavior. However, elucidating the molecular spatial organization of the cell-surface glycoproteome within the native cellular context has remained challenging due to its structural complexity and density. Here, we introduce a super-resolution microscopy-based approach that enables the spatial analysis of a protein and its glycosylation pattern directly in the cellular environment. Our strategy relies on a combination of lectin-based labeling, metabolic oligosaccharide engineering, and immunocytochemistry. This allows for the mapping of individual protein glycoforms as well as sialylation states of single proteins. By providing access to the spatial axis of the cell-surface glycoproteome, our technique opens up new avenues for understanding the molecular architecture of protein glycosylation in biological processes of central relevance, including, but not limited to, development, signaling, and immune system regulation.