Non-specific cysteine modifications on proteins induced by trans-cyclooctene.

Tetrazine-trans-cyclooctene (Tz-TCO) click chemistry has been widely adopted for various applications due to its faster reaction kinetics and higher biocompatibility compared to conventional copper-catalyzed click chemistry. However, most click chemistry groups do not appear to be fully bioorthogonal, as several of them-including tetrazine-have been found to directly modify proteins. Here, we report the unexpected discovery of non-specific protein modifications by TCO during the development of a tetrazine-based proximity labeling method. TCO probes readily conjugate to cysteine residues on proteins, and pre-treatment with iodoacetamide significantly reduces TCO-mediated background labeling. Chemoproteomic analyses reveal that proteins involved in translation are heavily modified by TCO, pointing to potential off-target effects when TCO probes are used in live cells. Our findings underscore the need for further improving the biocompatibility of click chemistry to enable more precise biological applications.