Proximity‐Induced Transfer of a Mass Tag Enables Direct Profiling of Active Matrix Metalloproteases

ABSTRACT Conventional activity‐based probes in activity‐based protein profiling (ABPP) require enrichment or reporter tags for detection, which limits sensitivity and multiplexing. Here, we present an enrichment‐free chemoproteomic approach that enables direct mass spectrometric detection by Matrix‐Assisted Laser Desorption/Ionization (MALDI) of active proteases. An active‐site–directed affinity probe transfers, through a proximity‐induced reaction, a MALDI‐detectable α‐cyano‐4‐hydroxycinnamic acid (CHCA) tag exclusively to catalytically active forms of matrix metalloproteases (MMPs). The CHCA label enhances ionization efficiency and markedly improves signal‐to‐noise ratios, allowing confident identification of CHCA‐labelled peptides under discriminating analytical conditions. Each active metalloprotease is thereby, associated with a distinct set of CHCA signature peptides, defining its activity fingerprint. This workflow achieves multiplexed and quantitative activity profiling of MMPs, directly in complex proteomes. This design expands ABPP into the mass spectrometry domain and establishes a robust platform for activity‐based enzyme detection.