Fluorescent labeling of live-cell surfaceome and its application in antibody-target interaction analysis

Cell-surface proteins play key roles in the communication between external stimuli and internal signaling. As protein types and expression levels vary in different cells, in-situ visualization of the whole surface proteome (surfaceome) may facilitate the study of their functions in homeostasis maintenance or response to environmental changes (e.g., drug treatment). However, there lacks easily-prepared and universal labeling probes to visualize them in living cells.

We designed and synthesized a small-molecule fluorescent probe, SRB-NHS, for one-step labeling of surfaceome. Live-cell imaging results exhibited the plasma membrane localization of the fluorescent signal from SRB-NHS and SDS-PAGE/fluorescence scanning results confirmed the covalent labeling of proteins by SRB-NHS, indicating the suitability of SRB-NHS for surfaceome labeling towards different cell lines.

Upon labeling by SRB-NHS, the cellular internalization of surfaceome was studied under different stimuli (e.g., nutritional deprivation, drug treatments). Intriguingly, specific monitoring of the interaction between antibody drugs and related cell-surface targets can be achieved when the probe is used in combination with fluorescently labeled antibodies and imaged via Förster resonance energy transfer (FRET), offering a new method compatible with various cell lines to monitor the surfaceome or a specific drug-target interaction in situ.