Abstract
In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin. Relative binding affinities of different peptides towards the protein could also be estimated using IF-MALDI-MS. This study may extend the application of IF-MALDI-MS in the analysis of noncovalent complexes and offer a perspective into the utility of MALDI-MS as an alternative approach to study the peptides binding to calmodulin.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Yamniuk, A. P.; Vogel, H. J. Calmodulin’s Flexibility Allows for Promiscuity in Its Interactions with Target Proteins and Peptides. Mol. Biotechnol. 2004, 27, 33–57.
Ishida, H.; Vogel, H. J. Protein-Peptide Interaction Studies Demonstrate the Versatility of Calmodulin Target Protein Binding. Protein Pept. Lett. 2006, 13, 455–465.
Brokx, R. D.; Lopez, M. M.; Vogel, H. J.; Makhatadze, G. I. Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding. J. Biol. Chem. 2001, 276, 14081–14091.
Comte, M.; Maulet, Y.; Cox, J. A. Ca2+-Dependent High-Affinity Complex Formation between Calmodulin and Melittin. Biochem. J. 1983, 209, 269–272.
Malencik, D. A.; Anderson, S. R. Binding of Simple Peptides, Hormones, and Neurotransmitters by Calmodulin. Biochemistry 1982, 21, 3480–3486.
Tanaka, Y.; Takahashi, S.; Mitsui, Y.; Itoh, S.; Iitaka, Y.; Kasai, H.; Okuyama, T. X-ray Crystallography and Chromatographic Characterization of the Crystals of Ca2+-Calmodulin Complexed with Bee Venom Melittin. J. Mol. Biol. 1985, 186, 675–677.
Kataoka, M.; Head, J. F.; Seaton, B. A.; Engelman, D. M. Melittin Binding Causes a Large Calcium-Dependent Conformational Change in Calmodulin. Proc. Nat. Acad. Sci. U.S.A. 1989, 86, 6944–6948.
Moorthy, A. K.; Gopal, B.; Satish, P. R.; Bhattacharya, S.; Bhattacharya, A.; Murthy, M. R. N.; Surolia, A. Thermodynamics of Target Peptide Recognition by Calmodulin and a Calmodulin Analogue: Implications for the Role of the Central Linker. FEBS Lett. 1999, 461, 19–24.
Li, W. G.; Cui, X. Q.; Yang, X. R.; Zhao, D. Q.; Ni, J. Z. Real-time Analysis of the Interaction between Calmodulin and Melittin by SPR Spectroscopy. Chin. Chem. Lett. 2002, 13, 165–166.
Papish, A. L.; Tari, L. W.; Vogel, H. L. Dynamic Light Scattering Study of Calmodulin-Target Peptide Complexes. Biophys. J. 2002, 83, 1455–1464.
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.
Calvo, E.; Camafeita, E.; Diaz, J. F.; Lopez, J. A. Mass Spectrometry for Studying the Interaction between Small Molecules and Proteins. Curr. Proteom. 2008, 5, 20–34.
Loo, J. A. Electrospray Ionization Mass Spectrometry: A Technology for Studying Noncovalent Macromolecular Complexes. Int. J. Mass Spectrom. 2000, 200, 175–186.
Nemirovskiy, O. V.; Ramanathan, R.; Gross, M. L. Investigation of Calcium-Induced, Noncovalent Association of Calmodulin with Melittin by Electrospray Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, 809–812.
Veenstra, T. D.; Tomlinson, A. J.; Benson, L.; Kumar, R.; Naylor, S. Low Temperature Aqueous Electrospray Ionization Mass Spectrometry of Noncovalent Complexes. J. Am. Soc. Mass Spectrom. 1998, 9, 580–584.
Mathur, S.; Badertscher, M.; Scott, M.; Zenobi, R. Critical evaluation of Mass Spectrometric Measurement of Dissociation Constants: Accuracy and Cross-Validation against Surface Plasmon Resonance and Circular Dichroism for the Calmodulin-Melittin System. Phys. Chem., Chem. Phys. 2007, 9, 6187–6198.
Nousiainen, M.; Derrick, P. J.; Lafitte, D.; Vainiotalo, P. Relative Affinity Constants by Electrospray Ionization and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Calmodulin Binding to Peptide Analogs of Myosin Light Chain Kinase. Biophys. J. 2003, 85, 491–500.
Nousiainen, M.; Feng, X. D.; Vainiotalo, P.; Derrick, P. J. Calmodulin-RS20-Ca4 Complex in the Gas Phase: Electrospray Ionisation and Fourier Transform Ion Cyclotron Resonance. Eur. J. Mass Spectrom. 2001, 7, 393–398.
Chevreux, G.; Potier, N.; Van Dorsselaer, A.; Bahloul, A.; Houdusse, A.; Wells, A.; Sweeney, H. L. Electrospray Ionization Mass Spectrometry Studies of Noncovalent Myosin VI Complexes Reveal a New Specific Calmodulin Binding Site. J. Am. Soc. Mass Spectrom. 2005, 16, 1367–1376.
Shirran, S.; Garnaud, P.; Daff, S.; McMillan, D.; Barran, P. The Formation of a Complex between Calmodulin and Neuronal Nitric Oxide Synthase is Determined by ESI-MS. J. R. Soc. Interface 2005, 2, 465–476.
Scaloni, A.; Miraglia, N.; Orru, S.; Amodeo, P.; Motta, A.; Marino, G.; Pucci, P. Topology of the Calmodulin-Melittin Complex. J. Mol. Biol. 1998, 277, 945–958.
Wong, J. W. H.; Maleknia, S. D.; Downard, K. M. Hydroxyl Radical Probe of the Calmodulin-Melittin Complex Interface by Electrospray Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2005, 16, 225–233.
Wattenberg, A.; Sobott, F.; Barth, H. D.; Brutschy, B. Studying Noncovalent Protein Complexes in Aqueous Solution with Laser Desorption Mass Spectrometry. Int. J. Mass Spectrom. 2000, 203, 49–57.
Bolbach, G. Matrix-Assisted Laser Desorption/Ionization Analysis of Noncovalent Complexes: Fundamentals and Applications. Curr. Pharm. Des. 2005, 11, 2535–2557.
Villanueva, J.; Yanes, O.; Querol, E.; Serrano, L.; Aviles, F. X. Identification of Protein Ligands in Complex Biological Samples Using Intensity-Fading MALDI-TOF Mass Spectrometry. Anal. Chem. 2003, 75, 3385–3395.
Yanes, O.; Villanueva, J.; Querol, E.; Aviles, F. X. Intensity-Fading MALDI-TOF MS: Novel Screening for Ligand Binding and Drug Discovery. Drug Discov. Today: TARGETS 2004, 3, 23–30.
Yanes, O.; Villanueva, J.; Querol, E.; Aviles, F. X. Detection of Noncovalent Protein Interactions by ‘Intensity Fading’ MALDI-TOF Mass Spectrometry: Applications to Proteases and Protease Inhibitors. Nature protocols 2007, 2, 119–130.
Kiselar, J. G.; Downard, K. M. Direct Identification of Protein Epitopes by Mass Spectrometry without Immobilization of Antibody and Isolation of Antibody-Peptide Complexes. Anal. Chem. 1999, 71, 1792–1801.
Kiselar, J. G.; Downard, K. M. Antigenic Surveillance of the Influenza Virus by Mass Spectrometry. Biochemistry 1999, 38, 14185–14191.
Kiselar, J. G.; Downard, K. M. Preservation and Detection of Specific Antibody-Peptide Complexes by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2000, 11, 746–750.
Downard, K. M.; Morrissey, B. Fingerprinting a Killer: Surveillance of the Influenza Virus by Mass Spectrometry. Analyst 2007, 132, 611–614.
Yanes, O.; Villanueva, J.; Querol, E.; Aviles, F. X. Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS. Mol. Cell. Proteom. 2005, 4, 1602–1613.
Yanes, O.; Nazabal, A.; Wenzel, R.; Zenobi, R.; Aviles, F. X. Detection of Noncovalent Complexes in Biological Samples by Intensity Fading and High-Mass Detection MALDI-TOF Mass Spectrometry. J. Proteom. Res. 2006, 5, 2711–2719.
Yanes, O.; Aviles, F. X.; Roepstorff, P.; Jorgensen, T. J. D. Exploring the “Intensity Fading” Phenomenon in the Study of Noncovalent Interactions by MALDI-TOF Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2007, 18, 359–367.
Sugaya, M.; Saito, R.; Matsumura, Y.; Harada, K.; Katoh, A. Facile Detection of Specific RNA-Polypeptide Interactions by MALDI-TOF Mass Spectrometry. J. Pept. Sci. 2008, 14, 978–983.
Shabab, M.; Kulkarni, M. J.; Islam Khan, M. Study of Papain-Cystatin Interaction by Intensity Fading MALDI-TOF-MS. Protein J. 2008, 27, 7–12.
Schlosser, G.; Pocsfalvi, G.; Malorni, A.; Puerta, A.; de Frutos, M.; Vekey, K. Detection of Immune Complexes by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 2741–2747.
Yoshino, H.; Wakitan, M.; Izumi, Y. Calcium-Dependent Changes in Structure of Calmodulin with Substance P. J. Biol. Chem. 1993, 268, 12123–12128.
Hu, P. F.; Loo, J. A. Determining Calcium-Binding Stoichiometry and Cooperativity of Parvalbumin and Calmodulin by Mass Spectrometry. J. Mass Spectrom. 1995, 30, 1076–1082.
Toubouly, D.; Brunelle, A.; Laprevote, O. Characterization of α-Parvalbumin on Muscle Tissue Sections by in situ Calcium Attachment. Rapid Commun. Mass Spectrom. 2007, 21, 3756–3758.
Zhu, M. M.; Rempel, D. L.; Zhao, J.; Giblin, D. E.; Gross, M. L. Probing Ca2+-Induced Conformational Changes in Porcine Calmodulin by H/D Exchange and ESI-MS: Effect of Cations and Ionic Strength. Biochemistry 2003, 42, 15388–15397.
Zehl, M.; Allmaier, G. Instrumental Parameters in the MALDI-TOF Mass Spectrometric Analysis of Quaternary Protein Structures. Anal. Chem. 2005, 77, 103–110.
Vogl, T.; Roth, J.; Sorg, C.; Hillenkamp, F.; Strupat, K. Calcium-Induced Noncovalently Linked Tetramers of MRP8 and MRP14 Detected by Ultraviolet Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1999, 10, 1124–1130.
Horneffer, V.; Strupata, K.; Hillenkamp, F. Localization of Noncovalent Complexes in MALDI-Preparations by CLSM. J. Am. Soc. Mass Spectrom. 2006, 17, 1599–1604.
Wortmann, A.; Pimenova, T.; Alves, S.; Zenobi, R. Investigation of the First Shot Phenomenon in MALDI Mass Spectrometry of Protein Complexes. Analyst 2007, 132, 199–207.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Published online November 27, 2008
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Wang, Z., Yu, X., Cui, M. et al. Investigation of calmodulin-peptide interactions using matrix-assisted laser desorption/ionization mass spectrometry. J Am Soc Mass Spectrom 20, 576–583 (2009). https://doi.org/10.1016/j.jasms.2008.11.017
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2008.11.017