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

ABSTRACT 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, Xenopus laevis , 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.