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

ABSTRACT

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.