Dual‐Organelle Secretome Conjugation and Organ‐Uptake Tracking (DuO‐SCOUT) Enables Deep and Sensitive Mapping of the Secreted Proteins

Inter-organ communication is governed by a complex "secretome," yet mapping the journey of these factors from their origin to precise cellular destinations remains a fundamental challenge. Proximity labeling emerges as a powerful tool to dissect the secretome, yet conventional workflows typically rely on single-compartment biotinylation at the endoplasmic reticulum (ER), failing to capture proteins that utilize unconventional secretion. Here, we present DuO-SCOUT (Dual-Organelle Secretome Conjugation and Organ-Uptake Tracking), a high-performance platform that simultaneously targets BioID2 to the ER and the trans-Golgi network (TGN). This integrated strategy captures the full secretory maturation relay, increasing protein identification by over 120% compared to traditional ER-anchored methods. We translated this in vivo using an Adipoq-Cre mouse model to map the adipose secretome. To bridge the gap between systemic transport and tissue-specific uptake, DuO-SCOUT integrates BSPA (Biotin-Specific Proximity Amplification), a visualization toolkit detecting biotinylated proteins with sub-nanomolar sensitivity. In obese mice, we identified the piriform cortex (PIR) as a previously unrecognized extra-hypothalamic sink for adipose-derived leptin. This is associated with a localized neuroinflammatory signature, including significant induction of Il6. DuO-SCOUT establishes a broadly applicable framework for dissecting the complex molecular logic of systemic organ-organ communication.