title: Elucidating the site of protein-ATP binding by top-down mass spectrometry.
abstract: A Fourier-transform ion cyclotron resonance (FT-ICR) top-down mass spectrometry strategy for determining the adenosine triphosphate (ATP)-binding site on chicken adenylate kinase is described. Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), but the ability to detect protein-ligand complexes depends on their stability in the gas phase. Previously, we showed that collisionally activated dissociation (CAD) of protein-nucleotide triphosphate complexes yield products from the dissociation of a covalent phosphate bond of the nucleotide with subsequent release of the nucleotide monophosphate (Yin, S. et al., J. Am. Soc. Mass Spectrom. 2008, 19, 1199-1208). The intrinsic stability of electrostatic interactions in the gas phase allows the diphosphate group to remain noncovalently bound to the protein. This feature is exploited to yield positional information on the site of ATP-binding on adenylate kinase. CAD and electron capture dissociation (ECD) of the adenylate kinase-ATP complex generate product ions bearing mono- and diphosphate groups from regions previously suggested as the ATP-binding pocket by NMR and crystallographic techniques. Top-down MS may be a viable tool to determine the ATP-binding sites on protein kinases and identify previously unknown protein kinases in a functional proteomics study.
authors.affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California 90095, USA.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yin
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D005583:Fourier Analysis; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D055672:Static Electricity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000255:Adenosine Triphosphate; D000263:Adenylate Kinase