A Single Probe for Two Separate Imaging Applications: Turn-On Labeling of the Cell-Surface Proteome and Wash-Free...

The plasma membrane is a dynamic interface essential for cellular homeostasis, yet there is a lack of a single, unified tool to monitor its integrity and track the dynamic reorganization of its protein landscape (the surfaceome) in a simple, real-time manner. Here, we report the design and application of CAZ-ONBD, a fluorogenic probe that enables wash-free, one-step covalent labeling of the live-cell surfaceome and acts as a real-time indicator of membrane damage. CAZ-ONBD combines a membrane-anchoring amphiphilic zwitterion (CAZ) with a fluorogenic O-nitrobenzoxazolone (O-NBD) module. Upon insertion into the plasma membrane, CAZ-ONBD undergoes a turn-on reaction with proximal lysine residues on surface proteins, enabling the background-free imaging of the surfaceome. Crucially, the probe is cell-impermeable under normal conditions. However, upon membrane compromise, it enters the cytosol and is activated by intracellular thiols (e.g., cysteine), generating a strong fluorescent signal that reports on damage. We validated the utility of CAZ-ONBD in multiple live-cell systems, demonstrating its ability to covalently label diverse cell types, track stimulus-induced protein internalization, and visualize membrane damage in real time. Furthermore, we successfully extended its application to label the surfaceome of live zebrafish embryos. This work provides a versatile chemical tool for integrating the real-time assessment of membrane integrity with the dynamic visualization of surface proteome trafficking, offering a powerful platform for deciphering cell stress responses and advancing drug discovery.