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.