In situ proximity labeling reveals the proteome and signaling landscape of presynaptic boutons

Current proteomic analyses of presynaptic compartments largely rely on synaptosome fractions prepared from whole brain or dissected brain regions. However, the accurate identification of proteins that are truly enriched at presynaptic sites is limited by the purification efficiency and the intrinsic heterogeneity of synaptosome preparations. Alternative approaches that achieve higher spatial specificity are therefore required. Here, we established an in situ proximity biotinylation platform based on APEX2 to profile the proteomes of presynaptic boutons and their neighboring axons in living neurons. In cultured hippocampal neurons, presynaptic boutons were selectively labeled using APEX2-Synapsin 1, whereas axonal regions including presynaptic boutons were labeled using APEX2-SNAP-25, enabling compartment-resolved proteomic comparison. Through quantitative mass spectrometry and systematic analysis, we selected approximately 500 proteins that are preferentially enriched in boutons relative to axons. This bouton-enriched proteome includes canonical synaptic vesicle proteins and well-characterized active zone components, validating the spatial specificity of the approach. In addition, it reveals protein clusters suggestive of specialized signaling pathways related to ATP production and protein degradation, indicating previously underappreciated metabolic and proteostatic specialization enriched in presynaptic terminals. Together, our dataset provides a resource for the study of synaptic function under physiological and pathological conditions and establishes a generalizable strategy for spatially resolved proteomic analysis of neuronal subcompartmentation.