Bioorthogonal Chemistry in Neurobiology: From Imaging to Proteomics.

Bioorthogonal click chemistry has emerged as a versatile approach for probing molecules with high selectivity and minimal perturbation. While applicable to diverse biomolecules, its use in protein studies has proven particularly powerful, especially in neurobiology, where precise molecular tools are essential. A key technology enabling such precision is genetic code expansion (GCE), which allows the incorporation of unnatural amino acids (UAAs) bearing bioorthogonal handles into proteins. These UAAs can be introduced site-specifically to label defined proteins or proteome-wide for systems-level analysis. Complementing this, proteome-wide labeling can be achieved through metabolic incorporation of amino acid analogs. Coupled with copper-catalyzed or strain-promoted click reactions, these labeling technologies enable applications ranging from protein visualization to mass spectrometry-based proteomics. Ongoing advances in UAA incorporation strategies and the chemical diversity of available UAAs continue to broaden the opportunities to study protein expression, localization, and dynamics. In this review, we highlight recent methodological developments in click chemistry-based protein labeling and their applications in neurobiology.