Chemoproteomic Profiling of ATP-Binding Sites with Acid-Cleavable Photoreactive Adenosine Phosphate Probes

Adenosine triphosphate (ATP) is a key molecule in various cellular processes. The human proteome contains over 1500 annotated ATP-binding proteins, many of which function through the conversion of ATP to adenosine diphosphate (ADP). Evaluating their binding patterns with different nucleotides would facilitate our understanding of their working mechanisms. We report here acid-cleavable photoreactive ATP and ADP probes for the global profiling of ATP- and ADP-binding proteins and their binding sites. These probes incorporate a diazirine photoreactive group for the covalent labeling and an alkyne handle for the imaging or enrichment of labeled proteins. The linkage between the modification groups and ATP or ADP would be cleaved under acidic conditions, releasing ATP or ADP from labeled peptides, which is beneficial for LC-MS/MS-based labeling-site identification. Both probes showed a good labeling efficiency for various ATP-binding proteins. Direct comparative analysis revealed distinct labeling-site preferences between the two probes and a more favorable labeling of RNA helicases by the ADP probe. These findings provided insights into the binding patterns of ATP and ADP, advancing our understanding of the functional mechanisms of related proteins. This method is a powerful tool for studying ATP-binding proteins and their functions in health and disease.