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

## TL;DR

This paper introduces a new method to detect active enzymes at the single-molecule level using probes, which could help identify enzyme activity in complex systems like blood.

## Contribution

The study presents a novel single-molecule enzyme activity assay using activity-based probes for serine hydrolases.

## Key findings

- The assay successfully detects active serine hydrolases like PSA and granzyme B in complex biological systems.
- Active granzyme B in blood may serve as a potential indicator of liver damage linked to immune cell activation.

## 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, 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

This study introduces a single‐molecule strategy using activity‐based probes (ABPs) to sensitively detect accessible enzymes in complex biological systems. The approach successfully visualizes the active forms of PSA and granzyme B, demonstrating its utility for probing protease activity states with high selectivity and sensitivity at the molecular level.