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 ATP-binding 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 MS(2)/MS(3) 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.
Renzone
Giovanni
G
Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy.
Salzano
Anna Maria
AM
Arena
Simona
S
D'Ambrosio
Chiara
C
Scaloni
Andrea
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D003839:Deoxyadenosines / Q000737:chemistry*; D006593:Hexokinase / Q000737:chemistry* / Q000378:metabolism; D007477:Ions / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000032:analysis*; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
D003839:Deoxyadenosines; D007477:Ions; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C063171:5'-fluorosulfonylbenzoyldeoxyadenosine; D000255:Adenosine Triphosphate; D006593:Hexokinase; D010735:Phosphoglycerate Kinase