Development of single-molecule enzyme activity assay for serine hydrolases using activity-based protein labeling probes

ABSTRACT 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, we report the development of single-molecule enzyme activity assays utilizing activity-based probes that label active enzymes in an enzyme superfamily-wide manner. A proof-of-principle using fluorophosphonate-based probes was 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 suggested the potential of active granzyme B in blood as a suitable biomarker of liver damage with specific activation of immune cells.