Clickable Photoreactive ATP-Affinity Probe for Global Profiling of ATP-Binding Proteins

Adenosine
triphosphate (ATP) is the major energy carrier in organisms,
and there are many cellular proteins that can bind to ATP. Among these
proteins, kinases are key regulators in several cell signaling processes,
and aberrant kinase signaling contributes to the development of many
human diseases, including cancer. Hence, small-molecule kinase inhibitors
have been successfully used for the treatment of various diseases.
Since the ATP-binding pockets are similar for many kinases, it is
very important to evaluate the selectivity of different kinase inhibitors.
We report here a clickable ATP photoaffinity probe for the global
profiling of ATP-binding proteins. After incubating the protein lysate
with the ATP probe followed by ultraviolet (UV) irradiation, ATP-binding
proteins were labeled with an alkyne handle for subsequent biotin
conjugation through click chemistry. Labeled proteins were enriched
with streptavidin beads, digested with trypsin, and analyzed using
liquid chromatography–tandem mass spectrometry (LC-MS/MS).
More than 400 ATP-binding proteins, including approximately 200 kinases,
could be identified in a single LC-MS/MS run in the data-dependent
acquisition mode. We then applied this method to the analysis of targets
of three selected ATP-competitive kinase inhibitors. We were able
to successfully identify some of their reported target proteins from
label-free quantification results and validated the results using
Western blot analyses. Together, we developed a clickable ATP photoaffinity
probe for proteome-wide profiling of ATP-binding proteins and demonstrated
that this chemoproteomic method is amenable to high-throughput target
identification of kinase inhibitors.