Interface-Resolved Proteomics of Cell–Cell Membranes Reveals Early Spatial Polarity in a Vertebrate Embryo

Cell-cell membrane interfaces are central sites of adhesion, signaling, and polarity establishment, yet they have remained inaccessible to proteome-wide analysis as discrete analytical units. Here, we report an interface-resolved proteomics workflow that isolates intact intercellular membrane segments from single, identified blastomeres and quantitatively profiles their protein composition. Using a microdissection-enabled strategy combined with optimized mild-detergent extraction and high-sensitivity high-resolution mass spectrometry, we achieve deep coverage of low-input membrane samples, identifying ∼3,000 proteins per interface type, including over 100 annotated plasma-membrane proteins. Applying this approach to defined dorsal-dorsal, dorsal-ventral, and ventral-ventral cell-cell interfaces in a 16-cell chordate embryo model, <i>Xenopus laevis</i> , reveals reproducible, interface-specific proteomic signatures that distinguish neighboring membrane contacts along the primary body axis. Region-enriched proteins include regulators of membrane trafficking, signaling, cytoskeletal organization, and metabolic pathways linked to early dorsal-ventral patterning. These results demonstrate that intercellular membrane interfaces exhibit molecular polarity at early developmental stages and establish interface-resolved proteomics as a general strategy for mapping spatially organized biochemical activities at cell-cell contacts.