TOF-probe-based mass cytometry reveals individual protease activity as an important driver of immune cell differentiation and function

Quantifying enzymatic activity at the single-cell level remains challenging. Building on previous development of metal-tagged activity-based probes (time-of-flight [TOF]-probes) compatible with mass cytometry, we adapt this technology to quantify individual proteases in immune cells. We optimize and validate TOF-probes targeting catalytically active cysteine and serine proteases in two primary immune populations: neutrophils and monocyte-derived macrophages. Using this platform, we observe striking shifts in cathepsin activity during monocyte-to-macrophage differentiation, along with a functional rewiring from apoptotic to pyroptotic protease programs. In neutrophils, unexpectedly, we detect high levels of active serine proteases even in the resting state, highlighting their potential role as pre-armed effector cells poised for rapid inflammatory responses. These findings lay the foundation for using TOF-probes to quantify active proteases in situ at the single-cell level and provide new insights into protease activation states across immune cells.