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Elucidating the site of protein-ATP binding by top-down mass spectrometry
1Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California 90095, USA.
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.
Insights
This study uses mass spectrometry to pinpoint adenosine triphosphate (ATP) binding sites on chicken adenylate kinase. The method leverages the stability of protein-ligand complexes in gas phase analysis for functional proteomics.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Proteomics
Background:
- Noncovalent protein-ligand complexes are challenging to detect using mass spectrometry due to gas-phase stability limitations.
- Previous work demonstrated that collisionally activated dissociation (CAD) of protein-nucleotide triphosphate complexes results in nucleotide monophosphate release, with the diphosphate remaining bound.
- Electrostatic interactions in the gas phase stabilize the diphosphate group's noncovalent attachment to the protein.
Purpose of the Study:
- To develop a Fourier-transform ion cyclotron resonance (FT-ICR) top-down mass spectrometry strategy for identifying the adenosine triphosphate (ATP)-binding site on chicken adenylate kinase.
- To utilize the gas-phase stability of protein-ligand complexes to gain positional information about ATP binding.
- To explore the potential of top-down mass spectrometry in functional proteomics for identifying protein kinase ATP-binding sites.
Main Methods:
- Employing Fourier-transform ion cyclotron resonance (FT-ICR) top-down mass spectrometry.
- Utilizing electrospray ionization mass spectrometry (ESI-MS) for detecting noncovalent protein-ligand complexes.
- Applying collisionally activated dissociation (CAD) and electron capture dissociation (ECD) to the adenylate kinase-ATP complex.
Main Results:
- The FT-ICR top-down mass spectrometry strategy successfully detected the adenylate kinase-ATP complex.
- CAD and ECD generated product ions containing mono- and diphosphate groups.
- These product ions mapped to regions previously identified as the ATP-binding pocket by NMR and crystallographic methods.
Conclusions:
- Top-down mass spectrometry is a viable approach for determining ATP-binding sites on proteins.
- This method can be applied to functional proteomics for identifying protein kinases and their binding sites.
- The strategy exploits the unique gas-phase stability of protein-nucleotide complexes.
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