Enhanced Membrane Preference in APEX2 Proximity Labeling via an iEDDA Strategy With Built‐In Background Correction

Proximity‐labeling techniques, such as engineered ascorbate peroxidase (APEX2), have revolutionized the mapping of spatially organized proteomes. However, achieving high spatial resolution and specificity remains challenging due to the diffusible nature of labeling intermediates and the difficulty in establishing robust negative controls. Here, we present a chemical strategy that addresses both challenges by engineering the labeling reporter itself. We developed a lipophilic APEX2 reporter, phenol‐ trans ‐cyclooctene (phenol‐TCO), designed to partition into membrane microenvironments. This reporter leverages inverse electron‐demand Diels–Alder (iEDDA) chemistry for subsequent detection. Crucially, we repurpose the inherent, non‐specific reactivity of the tetrazine–biotin conjugate to establish a powerful and practical built‐in negative control. When applied to profile the Lamin A‐associated proteome, the phenol‐TCO reporter enriches nuclear envelope and endoplasmic reticulum membrane proteins with high confidence, yielding a focused dataset. In sharp contrast, conventional phenol‐biotin labeled a diffuse set of proteins lacking lamina specificity. We demonstrate that the tetrazine‐biotin‐treated lysate control effectively removes non‐specific background while preserving genuine proximal interactors, supporting it as a practical alternative to traditional controls. This work utilizes reporter localization preference to enhance the spatial precision of proximity labeling and provides a generalizable control strategy, opening new avenues for dissecting protein networks with membrane emphasis.