A Click Chemistry-Based Biorthogonal Approach for the Detection and Identification of Protein Lysine Malonylation for Osteoarthritis Research.

Lysine malonylation is a post-translational modification in which a malonyl group, characterized by a negatively charged carboxylate, is covalently attached to the ε-amino side chain of lysine, influencing protein structure and function. Our laboratory identified Mak upregulation in cartilage under aging and obesity, contributing to osteoarthritis (OA). Current antibody-based detection methods face limitations in identifying Mak targets. Here, we introduce an alkyne-functionalized probe, MA-diyne, which metabolically incorporates into proteins, enabling copper-(I) ion-catalyzed click reactions to conjugate labeled proteins with azide-based fluorescent dyes or affinity purification tags. In-gel fluorescence confirms MA-diyne incorporation into proteins across various cell types and species, including mouse chondrocytes, adipocytes, HEK293T cells, and . Pull-down experiments identified known Mak proteins, such as GAPDH and Aldolase. The extent of MA-diyne modification was higher in Sirtuin 5-deficient cells, suggesting these modified proteins are Sirtuin 5 substrates. Pulse-chase experiments confirmed the dynamic nature of the protein malonylation. Quantitative proteomics identified 1136 proteins corresponding to 8903 peptides, with 429 proteins showing a 1-fold increase in the labeled group. Sirtuin 5 regulated 374 of these proteins. Pull down of newly identified proteins, such as β-actin and Stat3, was also done. This study highlights MA-diyne as a powerful chemical tool to investigate the molecular targets and functions of lysine malonylation under OA conditions.