SELECTIVE ION TRACING AND MSn ANALYSIS OF PEPTIDE DIGESTS FROM FSBA-TREATED KINASES FOR THE ANALYSIS OF PROTEIN ATP-BINDING SITES.

Kinases play a key role in many cellular processes by catalyzing the transfer of phosphoryl groups from ATP to a broad number of substrates, including amino acids on target proteins. The reagent 5¢-fluorosulfonylbenzoyl- 5¢-adenosine (FSBA) has been widely used to identify ATPbinding sites in kinases since it reacts with nucleophilic amino acids occurring within these motifs, determining a mass increase of 433 Da. In this study, we present a versatile MS approach that has been developed to recognize labeled peptides generated after enzymatic digestion of FSBA-treated kinases. Using selective ion tracing and MS2/MS3 experiments, we were able to easily identify peptides occurring at protein ATP-binding sites, also affording a complete characterization of the modified amino acids. This methodology may be used in the development of future parent ion scanning-based applications directed to large scale analysis of kinases within complex protein mixtures.