Localized Hydrolysis‐Based Proximity Reporting Platform for Protein Hierarchical Structure Profiling

Abstract The substrate‐specific bond cleavage capability of hydrolases provides a unique strategy for engineering off‐on switches. This prompted our hypothesis that confining hydrolases and substrates to distinct biomolecular hierarchies (e.g., a protein and its modifying glycans) could enable transmission of spatial proximity information through localized hydrolysis. Using New Delhi metallo‐β‐lactamase 1 (NDM‐1), we developed a localized hydrolysis‐based proximity reporting (LHPR) platform for in situ elucidation of the hierarchical architecture of glycoproteins. We constructed protein probes by conjugating target‐recognizing molecules with NDM‐1, and designed click chemistry‐reactive, substrate‐caged fluorescent molecules as glycan probes. Programmed NDM‐1 activity enabled in situ protein‐specific imaging of glycans via proximity‐dependent activation within various biological specimens. Within a unified enzymatic framework, we successfully quantified distinct glycans of individual proteins and established the principle of protein‐specific glycosylation stoichiometry analysis. Our work provides a powerful tool for developing glycoprotein‐based disease biomarkers, therapeutic targets, and diagnostic approaches, offering new possibilities for precision medicine research.