Abstract
It was shown in previous work that the interaction of growth factors (GFs) with adenosine triphosphate (ATP) is essential for their neuroprotective effect. Here we investigated the nature of the association of human basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) with ATP. It was demonstrated that this interaction involves the formation of non-covalent ATP-GF complexes that are labile at low pH and that could be selectively purified and subjected to electrospray and MALDI-TOF mass spectrometry. The results obtained with these techniques indicated that the stability of the complexes is high. Main features of the procedure used here are: (1) reversed-phase purification of nucleotide-protein non-covalent complexes, (2) their detection with MALDI-TOF-MS using acid-free matrix, and/or (3) their measurement with ESI-MS using soft desolvation conditions. The methodology was successful in providing proof for the presence of various nucleotide-GF complexes. It was extended to other nucleotide-binding proteins (ribonuclease A) as well as proteins that do not exhibit nucleotide binding (lysozyme) as positive and negative control, respectively. Thus, the method demonstrated its general use for the investigation of a wide range of proteins interacting with nucleotides as long as their complexes are sufficiently stable to accommodate the experimental conditions.
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Klumpp, S.; Kriha, D.; Bechmann, G.; Maaßen, A.; Maier, S.; Pallast, S.; Hoell, P.; Krieglstein, J. Phosphorylation of the Growth Factors bFGF, NGF and BDNF: A Prerequisite for Their Biological Activity. Neurochem. Int 2006, 48, 131–137.
Napper, S.; Kindrachuk, J.; Olson, D. J.; Ambrose, S. J.; Dereniwsky, C.; Ross, A. R. Selective Extraction and Characterization of a Histidine-Phosphorylated Peptide Using Immobilized Copper(II) Ion Affinity Chromatography and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Anal. Chem 2003, 75, 1741–1747.
Medzihradszky, K. F.; Phillipps, N. J.; Senderowicz, L.; Wang, P.; Turck, C. W. Synthesis and Characterization of Histidine-Phosphorylated Peptides. Protein Sci 1997, 6, 1405–1411.
Wind, M.; Wegener, A.; Kellner, R.; Lehmann, W. D. Analysis of CheA Histidine Phosphorylation and Its Influence on Protein Stability by High-Resolution Element and Electrospray Mass Spectrometry. Anal. Chem 2005, 77, 1957–1962.
Nosworthy, N. J.; Peterkofsky, A.; König, S.; Seok, Y. J.; Szczepanowski, R. H.; Ginsburg, A. Phosphorylation Destabilizes the Amino-Terminal Domain of Enzyme I of the Escherichia coli Phosphenolpyruvate:Sugar Phosphotransferase System. Biochemistry 1998, 37, 6718–6726.
Collet, J. F.; Stoobant, V.; Van Schaftingen, E. Mechanistic Studies of Phosphoserine Phosphatase, an Enzyme Related to P-type ATPases. J. Biol. Chem 1999, 274, 33985–33990.
Sobott, F.; McCammon, M. G.; Hernandez, H.; Robinson, C. V. The Flight of Macromolecular Complexes in a Mass Spectrometer. Philos. Trans. R. Soc. Lond. A 2005, 363, 379–391.
Camilleri, P.; Haskins, N. J. Investigating the Noncovalent Interaction of Cytidylic Acids with Ribonuclease A by Electrospray Mass-Spectrometry. Rapid Commun. Mass Spectrom 1993, 7, 603–604.
Pinkse, 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.
Huang, E. C.; Pramanik, B. N.; Tsarbopoulos, A.; Reichert, P.; Ganguly, A. K.; Trotta, P. P.; Nagabhushan, T. L. Application of Electrospray Mass Spectrometry in Probing Protein-Protein and Protein-Ligand Noncovalent Interactions. J. Am. Soc. Mass Spectrom 1993, 4, 624–630.
Ganguly, A. K.; Pramanik, B. N.; Tsarbopoulos, A.; Covey, T. R.; Huang, E.; Fuhrman, S. A. Mass-Spectrometric Detection of the Noncovalent Gdp-Bound Conformational State of the Human H-Ras Protein. J. Am. Chem. Soc 1992, 114, 6559–6560.
König, S.; Hasche, A.; Pallast, S.; Krieglstein, J.; Klumpp, S. Proceedings of the 40th Annual Conference of the German Society of Mass Spectrometry, Bremen, March 11–14, 2007.
Rattenholl, A.; Lilie, H.; Grossmann, A.; Stern, A.; Schwarz, E.; Rudolph, R. The Pro-Sequence Facilitates Folding of Human Nerve Growth Factor from Escherichia coli Inclusion Bodies. Eur. J. Biochem 2001, 268, 3296–3303.
König, S.; Fales, H. M.; Haegele, K. D. Comment on the Cylindrical Capacitor Electrospray Interface. Anal. Chem 1998, 70, 4453–4455.
Pitteri, S. J.; Chrisman, P. A.; Badman, E. R.; McLuckey, S. A. Charge-State Dependent Dissociation of a Trypsin/Inhibitor Complex via Ion Trap Collisional Activation. Int. J. Mass Spectrom 2006, 253, 147–155.
Loboda, A. V.; Ackloo, S.; Chernushevich, I. V. A High-Performance Matrix-Assisted Laser Desorption/Ionization Orthogonal Time-of-Flight Mass Spectrometer with Collisional Cooling. Rapid Commun. Mass Spectrom 2003, 17, 2508–2516.
Wang, Y.; Schubert, M.; Ingendoh, A.; Franzen, J. Analysis of Non-Covalent Protein Complexes up to 290 kDa Using Electrospray Ionization and Ion Trap Mass Spectrometry. Rapid Commun. Mass Spectrom 2000, 14, 12–17.
Bothwell, M. A.; Shooter, E. M. Dissociation Equilibrium Constant of Beta Nerve Growth Factor. J. Biol. Chem 1977, 252, 8532–8536.
Kastrup, J. S.; Eriksson, E. S.; Dalboge, H.; Flodgaard, H. X-ray Structure of the 154-Amino-acid Form of Recombinant Human Basic Fibroblast Growth Factor: Comparison with the Truncated 146-Amino-Acid Form. Acta Cryst 1997, D53, 160–168.
McDonald, N. Q.; Lapatto, R.; Murray-Rust, J.; Gunning, J.; Wlodawer, A.; Blundell, T. L. New Protein Fold Revealed by a 2.3-Å Resolution Crystal Structure of Nerve Growth Factor. Nature 1991, 354, 411–414.
Kumar, K.; Jenkins, J. L.; Jardine, A. M.; Shapiro, R. Inhibition of Mammalian Ribonuclases by Endogenous Adenosine Dinucleotides. Biochem. Biophys. Res. Commun 2003, 300, 81–86.
Wetter, L. R.; Deutsch, H. F. Immunological Studies on Egg White Proteins. J. Biol. Chem 1951, 192, 237–242.
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Published online October 30, 2007
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König, S., Hasche, A., Pallast, S. et al. Detection of ATP-binding to growth factors. J. Am. Soc. Spectrom. 19, 91–95 (2008). https://doi.org/10.1016/j.jasms.2007.10.010
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DOI: https://doi.org/10.1016/j.jasms.2007.10.010