Abstract
A method to separate specific and nonspecific noncovalent interactions observed in ESI mass spectra between a protein and its ligands is presented. Assuming noncooperative binding, the specific ligand binding is modeled as a statistical distribution on identical binding sites. For the nonspecific fraction we assume a statistical distribution on a large number of “nonspecific” interacting sites. The model was successfully applied to the noncovalent interaction between the protein creatine kinase (CK) and its ligands adenosine diphosphate (ADP) and adenosine triphosphate (ATP) that both exhibit nonspecific binding in the mass spectrum. The two sequential dissociation constants obtained by applying our method are K1,diss=11.8±1.5µM and K2,diss = 48±6µM for ADP. For ATP, the constants are K1,diss = 27±7µM and K2,diss = 114±27µM. All constants are in good correlation with reported literature values. The model should be valuable for systems with a large dissociation constant that require high ligand concentrations and thus have increased potential of forming nonspecific adducts.
Article PDF
Similar content being viewed by others
References
Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray Ionization for Mass Spectrometry of Large Biomolecules. Science. 1989, 246, 464–471.
Loo, J. A. Electrospray Ionization Mass Spectrometry: A Technology for Studying Noncovalent Macromolecular Complexes. Int. J. Mass. Spectrom. 2000, 200, 175–186.
Veenstra, T. D. Electrospray Ionization Mass Spectrometry in the Study of Biomolecular Noncovalent Interactions. Biophys. Chem. 1999, 79, 63–79.
Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Quantitative Determination of Noncovalent Binding Interactions Using Soft Ionization Mass Spectrometry. Int. J. Mass Spectrom. 2002, 216, 1–27.
Smith, R. D. Evolution of ESI-Mass Spectrometry and Fourier Transform Ion Cyclotron Resonance for Proteomics and Other Biological Applications. Int. J. Mass Spectrom. 2000, 200, 509–544.
Heck, A. J. R.; Jørgensen, T. J. D. Vancomycin in Vacuo. Int. J. Mass Spectrom. 2004, 236, 11–23.
Robinson, C. V.; Chung, E. W.; Kragelund, B. B.; Knudsen, J.; Aplin, R. T.; Poulsen, F. M.; Dobson, C. M. Probing the Nature of Noncovalent Interactions by Mass Spectrometry. A Study of Protein-CoA Ligand Binding and Assembly. J. Am. Chem. Soc. 1996, 118, 8646–8653.
Peschke, M.; Verkerk, U. H.; Kebarle, P. Features of the ESI Mechanism that Affect the Observation of Multiply Charged Noncovalent Protein Complexes and the Determination of the Association Constant by the Titration Method. J. Am. Soc. Mass Spectrom. 2004, 15, 1424–1434.
Wang, W.; Kitova, E. N.; Klassen, J. S. Determination of Protein-Oligosaccharide Binding by Nanoelectrospray Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry. Methods Enzymol. 2003, 362, 376–396.
Wang, W.; Kitova, E. N.; Klassen, J. S. Nonspecific Protein-Carbohydrate Complexes Produced by Nanoelectrospray Ionization. Factors Influencing Their Formation and Stability. Anal. Chem. 2005, 77, 3060–3071.
Wang, W.; Kitova, E. N.; Sun, J.; Klassen, J. S. Blackbody Infrared Radiative Dissociation of Nonspecific Protein-Carbohydrate Complexes Produced by Nanoelectrospray Ionization: The Nature of the Noncovalent Interactions. J. Am. Soc. Mass Spectrom. 2005, 16, 1583–1594.
Pinske, M. W. H.; Heck, A. J. R.; Rumpel, K.; Pullen, F. Probing Noncovalent Protein-Ligand Interactions of the cGMP-Dependent Protein Kinase Using Electrospray Ionization Time of Flight Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2004, 15, 1392–1399.
Sundquist, G.; Benkestock, K.; Roeraade, J. Investigation of Multiple Binding Sites on Ribonuclease A Using Nano-Electrospray Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2005, 19, 1011–1016.
Benkestock, K.; Edlund, P.-O.; Roeraade, J. Electrospray Ionization Mass Spectrometry as a Tool for Determination of Drug Binding Sites to Human Serum Albumin by Noncovalent Interaction. Rapid Commun. Mass Spectrom. 2005, 19, 1637–1643.
Watts, D. C. Creatine Kinase (Adenosine 5′-Triphosphate-Creatine Phosphotransferase). In The Enzymes, Vol. VIII; Boyer, P. D., Ed.; Academic Press: New York/London, 1973; pp 383–455.
Borders, C. L.; Snider, M. J.; Wolfenden, R.; Edminston, P. L. Determination of the Affinity of Each Component of a Composite Quaternary Transition-State Analogue Complex of Creatine Kinase. Biochemistry. 2002, 41, 6995–7000.
McLaughlin, A. C. The Interaction of 8-Anilino-1-Nnaphtalenesulfonate with Creatine Kinase. J. Biol. Chem. 1974, 249(5), 1445–1452.
Burbaum, J. J.; Knowles, J. R. Internal Thermodynamics of Enzymes Determined by Equilibrium Quench: Values of Kint for Enolase and Creatine Kinase. Biochemistry. 1989, 28, 9306–9317.
Hornemann, T.; Rutishauser, D.; Wallimann, T. Why is Creatine Kinase a Dimer? Evidence for Cooperativity Between the Two Subunits. Biochim. Biophys. Acta. 2000, 1480(1/2), 365–373.
Kuby, S. A.; Mahowald, T. A.; Noltmann, E. A. Studies on Adenosine Triphosphate Transphosphorylases. IV. Enzyme-Substrate Interactions. Biochemistry. 1962, 1, 748–762.
Loo, J. A.; Ogorzalek Loo, R. G. Electrospray Ionization Mass Spectrometry of Peptides and Proteins. In Electrospray Ionization Mass Spectrometry. Cole, R. B., ed. John Wiley and Sons, Inc: New York, 1997, pp 385–419.
Chowdhury, S. K.; Katta, V.; Chait, B. T. Probing Conformational Changes in Proteins by Mass Spectrometry. J. Am. Chem. Soc. 1990, 112, 9012–9013.
Loo, J. A.; Hu, P.; McConnell, P.; Mueller, W. T.; Sawyer, T. K.; Thanabal, V. A Study of Src SH2 Domain Protein-Phosphopeptide Binding Interactions by Electrospray Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, 234–243.
Greig, M. J.; Gaus, H.; Cummins, L. L.; Sasmor, H.; Griffey, R. H. Measurement of Macromolecular Binding Using Electrospray Mass Spectrometry. Determination of Dissociation Constants for Oligonucleotide-Serum Albumin Complexes. J. Am. Chem. Soc. 1995, 117, 10765–10766.
Gabelica, V.; de Pauw, E.; Rosu, F. Interaction between Antitumor Drugs and a Double-Stranded Oligonucleotide Studied by Electrospray Ionization Mass Spectrometry. J. Mass Spectrom. 1999, 34, 1328–1337.
Jurchen, J. J.; Garcia, D. E.; Williams, E. R. Further Studies on the Origins of Asymmetric Charge Partitioning in Protein Homodimers. J. Am. Soc. Mass Spectrom. 2004, 15, 1408–1415.
Forstner, M.; Kriechbaum, M.; Laggner, P.; Wallimann, T. Structural Changes of Creatine Kinase Upon Substrate Binding. Biophys. J. 1998, 75, 1016–1023.
Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. Molecular Beams of Macro-Ions. J. Chem. Phys. 1968, 49, 2240–2249.
Kebarle, P.; Ho, Y. On the Mechanism of Electrospray Mass Spectrometry. In Electrospray Ionization Mass Spectrometry. Cole, R. B., ed. John Wiley and Sons, Inc.: New York, 1997, pp. 3–63.
Tanford, C. Multiple Equilibria. In Physical Chemistry of Macromolecules. Tanford, C., ed. John Wiley and Sons, Inc.: New York/London, 1961, pp. 526–586.
Author information
Authors and Affiliations
Additional information
Published online June 21, 2006
Rights and permissions
About this article
Cite this article
Daubenfeld, T., Bouin, AP. & van der Rest, G. A deconvolution method for the separation of specific versus nonspecific interactions in noncovalent protein-ligand complexes analyzed by ESI-FT-ICR mass spectrometry. The official journal of The American Society for Mass Spectrometry 17, 1239–1248 (2006). https://doi.org/10.1016/j.jasms.2006.05.005
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2006.05.005