Development of Single‐Molecule Enzyme Activity Assay for Serine Hydrolases Using Activity‐Based Protein Labeling Probes

Single-molecule enzyme activity assays have proven their potential in elucidating aberrant protein function at the proteoform level. However, the limited number of targetable enzymes is the major drawback of such assays. Here, the development of single-molecule enzyme activity assays utilizes activity-based probes that label active enzymes in an enzyme superfamily-wide manner. A proof-of-principle using fluorophosphonate-based probes is conducted to detect the active form of serine hydrolases such as PSA and granzyme B at the single-molecule level in complex biological systems such as blood. The assay suggests that active granzyme B in blood may serve as an indicator of liver damage associated with immune cell activation.