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A study of Src SH2 domain protein-phosphopeptide binding interactions by electrospray ionization mass spectrometry

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Journal of the American Society for Mass Spectrometry

Abstract

The noncovalent binding of various peptide ligands to pp60src (Src) SH2 (Src homology 2) domain protein (12.9 ku) has been used as a model system for development of electrospray ionization mass spectrometry (ESI-MS) as a tool to study noncovalently bound complexes. SH2 motifs in proteins are critical in the signal transduction pathways of the tyrosine kinase growth factor receptors and recognize phosphotyrosine-containing proteins and peptides. ESI-MS with a magnetic sector instrument and array detection has been used to detect the protein-peptide complex with low-picomole sensitivity. The relative abundances of the multiply charged ions for the complex formed between Src SH2 protein and several nonphosphorylated and phosphorylated peptides have been compared. The mass spectrometry data correlate well to the measured binding constants derived from solution-based methods, indicating that the mass spectrometry-based method can be used to assess the affinity of such interactions. Solution-phase equilibrium constants may be determined by measuring the amount of bound and unbound species as a function of concentration for construction of a Scatchard graph. ESI-MS of a solution containing Src SH2 with a mixture of phosphopeptides showed the expected protein-phosphopeptide complex as the dominant species in the mass spectrum, demonstrating the method’s potential for screening mixtures from peptide libraries.

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Author notes

  1. Peifeng Hu

    Present address: Baxter Healthcare Corporation, Route 120 & Wilson Road, WG3-15, 60073, Round Lake, IL

Authors and Affiliations

  1. Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan, USA

    Joseph A. Loo, Peifeng Hu, Patrick McConnell, W. Tom Mueller, Tomi K. Sawyer & Venkataraman Thanabal

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  1. Joseph A. Loo
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  2. Peifeng Hu
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  3. Patrick McConnell
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  4. W. Tom Mueller
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  6. Venkataraman Thanabal
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Loo, J.A., Hu, P., McConnell, P. et al. A study of Src SH2 domain protein-phosphopeptide binding interactions by electrospray ionization mass spectrometry. J Am Soc Mass Spectrom 8, 234–243 (1997). https://doi.org/10.1016/S1044-0305(96)00238-3

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  • DOI: https://doi.org/10.1016/S1044-0305(96)00238-3

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