Integration of Chemical Probes and Degradomics Reveals That Cathepsin B Functions Primarily as a Dipeptidyl Carboxypeptidase in Macrophages

Cathepsin B (Ctsb) is a lysosomal cysteine protease implicated in proteostasis and disease, but its endogenous substrate specificity and activity in intact cells remain incompletely defined. Here, we applied an integrated chemical biology and degradomics approach to profile Ctsb function in RAW264.7 macrophages using the irreversible inhibitor Z‐RK‐AOMK. Z‐RK‐AOMK treatment effectively inhibited intracellular Ctsb activity while also perturbing the broader cysteine cathepsin network, including altered activities of Ctsx and Ctsl, and accumulation of an active higher molecular weight Ctss species consistent with altered Ctss maturation. Despite these changes in cysteine cathepsin activity, Z‐RK‐AOMK treatment produced limited changes in global protein abundance and N‐terminal processing. In contrast, C‐terminomic profiling revealed enrichment of cleavage events occurring two and four residues upstream of native protein C‐termini. This pattern supports a predominant role for Ctsb as a dipeptidyl carboxypeptidase in cells and highlights C‐terminome profiling as a sensitive functional readout of its activity. Finally, we developed a fluorescent activity‐based probe, BODIPY‐RK‐AOMK, which selectively labels active Ctsb in cell lysates and live cells, enabling visualization of its predominantly lysosomal localization. Together, these findings define the cellular processing signature of Ctsb in macrophages and establish a complementary activity‐based probe for mapping its activity in biological systems.