A SPAAC-Based Bioorthogonal Method for Verifying Protein Palmitoylation

Palmitoylation is a crucial post-translational modification, and bioorthogonal chemistry based on azide-alkyne cycloaddition is typically used to verify protein palmitoylation. Traditional copper-catalyzed click chemistry (CuAAC) proceeds with fast kinetics and is widely used, but it requires a copper catalyst and suffers from copper-induced toxicity and nonspecific labeling. By contrast, strain-promoted click chemistry (SPAAC) has slower kinetics but is catalyst-free, offering high specificity, low cytotoxicity, and simple operation. However, SPAAC is mostly applied to live-cell labeling and imaging of known palmitoylated proteins in the field of palmitoylation, and its use in identifying novel palmitoylated proteins is still limited. Here, we present a SPAAC-based method for detecting endogenous protein palmitoylation. Compared with CuAAC, this method eliminates the need for copper catalysts and reducing agents, thereby simplifying the procedure and reducing reagent usage. Key features • A bioorthogonal method for identifying endogenous protein palmitoylation in cells. • Metabolical labeling of palmitoylated proteins in cells using azido palmitic acid. • The method requires simple steps and minimal reagents, reducing hands-on time and cost.