Using Peptide Array to Identify Binding Motifs and Interaction Networks for Modular Domains

  • Shawn S.-C. Li
  • Chenggang Wu
Part of the Methods in Molecular Biology™ book series (MIMB, volume 570)


Specific protein–protein interactions underlie all essential biological processes and form the basis of cellular signal transduction. The recognition of a short, linear peptide sequence in one protein by a modular domain in another represents a common theme of macromolecular recognition in cells, and the importance of this mode of protein–protein interaction is highlighted by the large number of peptide-binding domains encoded by the human genome. This phenomenon also provides a unique opportunity to identify protein–protein binding events using peptide arrays and complementary biochemical assays. Accordingly, high-density peptide array has emerged as a useful tool by which to map domain-mediated protein–protein interaction networks at the proteome level. Using the Src-homology 2 (SH2) and 3 (SH3) domains as examples, we describe the application of oriented peptide array libraries in uncovering specific motifs recognized by an SH2 domain and the use of high-density peptide arrays in identifying interaction networks mediated by the SH3 domain. Methods reviewed here could also be applied to other modular domains, including catalytic domains, that recognize linear peptide sequences.

Key words

Peptide arrays oriented peptide array library (or OPAL) SPOT celluspots domains src-homology 2 (or SH2) src-homology 3 (SH3) specificity linear motif protein–protein interaction (or PPI) interaction network 


  1. 1.
    Merrifield, R.B., (1963) Solid-phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc, 85, 2149–54.CrossRefGoogle Scholar
  2. 2.
    Frank, R. and Overwin, H., (1996) SPOT-synthesis epitope analysis with arrays of synthetic peptides prepared on cellulose membranes, Methods Mol Biol, 66, 149–169.PubMedGoogle Scholar
  3. 3.
    Wegner, G.J., Lee, H.J., and Corn, R.M., (2002) Characterization and optimization of peptide arrays for the study of epitope–antibody interactions using surface plasmon resonance imaging. Anal Chem, 74, 5161–8.PubMedCrossRefGoogle Scholar
  4. 4.
    Jia, C.Y., Nie, J., Wu, C., Li, C., and Li, S.S., (2005) Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Pro-rich region. Mol Cell Proteomics, 4, 1155–66.PubMedCrossRefGoogle Scholar
  5. 5.
    Rodriguez, M., Li, S.S., Harper, J.W., and Songyang, Z., (2004) An oriented peptide array library (OPAL) strategy to study protein–protein interactions. J Biol Chem, 279, 8802–7.PubMedCrossRefGoogle Scholar
  6. 6.
    Huang, H., Li, L., Wu, C., Schibli, D., Colwill, K., Ma, S., et al., (2008) Defining the specificity space of the human SRC homology 2 domain. Mol Cell Proteomics, 7, 768–84.PubMedGoogle Scholar
  7. 7.
    Frank, R., (1992) Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron, 48, 9217–32.CrossRefGoogle Scholar
  8. 8.
    Beyer, M., Nesterov, A., Block, I., Konig, K., Felgenhauer, T., Fernandez, S., et al., (2007) Combinatorial synthesis of peptide arrays onto a microchip. Science, 318, 1888.PubMedCrossRefGoogle Scholar
  9. 9.
    Pellois, J.P., Zhou, X., Srivannavit, O., Zhou, T., Gulari, E., and Gao, X., (2002) Individually addressable parallel peptide synthesis on microchips. Nat Biotechnol, 20, 922–6.PubMedCrossRefGoogle Scholar
  10. 10.
    Shin, D.S., Kim, D.H., Chung, W.J., and Lee, Y.S., (2005) Combinatorial solid phase peptide synthesis and bioassays. J Biochem Mol Biol, 38, 517–25.PubMedCrossRefGoogle Scholar
  11. 11.
    Falsey, J.R., Renil, M., Park, S., Li, S., and Lam, K.S., (2001) Peptide and small molecule microarray for high throughput cell adhesion and functional assays. Bioconjug Chem, 12, 346–53.PubMedCrossRefGoogle Scholar
  12. 12.
    Takahashi, M., Nokihara, K. and Mihara, H., (2003) Construction of a protein-detection system using a loop peptide library with a fluorescence label. Chem Biol, 10, 53–60.PubMedCrossRefGoogle Scholar
  13. 13.
    Salisbury, C.M., Maly, D.J., and Ellman, J.A., (2002) Peptide microarrays for the determination of protease substrate specificity. J Am Chem Soc, 124, 14868–70.PubMedCrossRefGoogle Scholar
  14. 14.
    Lesaicherre, M.L., Uttamchandani, M., Chen, G.Y., and Yao, S.Q., (2002) Developing site-specific immobilization strategies of peptides in a microarray. Bioorg Med Chem Lett, 12, 2079–83.PubMedCrossRefGoogle Scholar
  15. 15.
    Schultz, J., Hoffmuller, U., Krause, G., Ashurst, J., Macias, M.J., Schmieder, P., et al., (1998) Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels. Nat Struct Biol, 5, 19–24.PubMedCrossRefGoogle Scholar
  16. 16.
    Cestra, G., Castagnoli, L., Dente, L., Minenkova, O., Petrelli, A., Migone, N., et al., (1999) The SH3 domains of endophilin and amphiphysin bind to the proline-rich region of synaptojanin 1 at distinct sites that display an unconventional binding specificity. J Biol Chem, 274, 32001–7.PubMedCrossRefGoogle Scholar
  17. 17.
    Knoblauch, N.T., Rudiger, S., Schonfeld, H.J., Driessen, A.J., Schneider-Mergener, J., and Bukau, B., (1999) Substrate specificity of the SecB chaperone. J Biol Chem, 274, 34219–25.PubMedCrossRefGoogle Scholar
  18. 18.
    Pullen, S.S., Labadia, M.E., Ingraham, R.H., McWhirter, S.M., Everdeen, D.S., Alber, T., et al., (1999) High-affinity interactions of tumor necrosis factor receptor-associated factors (TRAFs) and CD40 require TRAF trimerization and CD40 multimerization. Biochemistry, 38, 10168–77.PubMedCrossRefGoogle Scholar
  19. 19.
    Landgraf, C., Panni, S., Montecchi-Palazzi, L., Castagnoli, L., Schneider-Mergener, J., Volkmer-Engert, R., et al., (2004) Protein interaction networks by proteome peptide scanning. PLoS Biol, 2, E14.PubMedCrossRefGoogle Scholar
  20. 20.
    Smith, M.J., Hardy, W.R., Murphy, J.M., Jones, N., and Pawson, T., (2006) Screening for PTB domain binding partners and ligand specificity using proteome-derived NPXY peptide arrays. Mol Cell Biol, 26, 8461–74.PubMedCrossRefGoogle Scholar
  21. 21.
    Wu, C., Ma, M.H., Brown, K.R., Geisler, M., Li, L., Tzeng, E., et al., (2007) Systematic identification of SH3 domain-mediated human protein–protein interactions by peptide array target screening. Proteomics, 7, 1775–85.PubMedCrossRefGoogle Scholar
  22. 22.
    Weiler, J., Gausepohl, H., Hauser, N., Jensen, O.N., and Hoheisel, J.D., (1997) Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays. Nucleic Acids Res, 25, 2792–9.PubMedCrossRefGoogle Scholar
  23. 23.
    Reineke, U., Kramer, A., and Schneider-Mergener, J., (1999) Antigen sequence- and library-based mapping of linear and discontinuous protein–protein-interaction sites by spot synthesis. Curr Top Microbiol Immunol, 243, 23–36.PubMedCrossRefGoogle Scholar
  24. 24.
    Buss, H., Dorrie, A., Schmitz, M.L., Frank, R., Livingstone, M., Resch, K., et al., (2004) Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity. J Biol Chem, 279, 49571–4.PubMedCrossRefGoogle Scholar
  25. 25.
    Szallasi, Z., Denning, M.F., Chang, E.Y., Rivera, J., Yuspa, S.H., Lehel, C., et al., (1995) Development of a rapid approach to identification of tyrosine phosphorylation sites: application to PKC delta phosphorylated upon activation of the high affinity receptor for IgE in rat basophilic leukemia cells. Biochem Biophys Res Commun, 214, 888–94.PubMedCrossRefGoogle Scholar
  26. 26.
    Espanel, X., Huguenin-Reggiani, M., and Van Huijsduijnen, R.H., (2002) The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities. Protein Sci, 11, 2326–34.PubMedCrossRefGoogle Scholar
  27. 27.
    Sparks, A.B., Rider, J.E., and Kay, B.K., (1998) Mapping the specificity of SH3 domains with phage-displayed random-peptide libraries. Methods Mol Biol, 84, 87–103.PubMedGoogle Scholar
  28. 28.
    Songyang, Z., Shoelson, S.E., McGlade, J., Olivier, P., Pawson, T., Bustelo, X.R., et al., (1994) Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol, 14, 2777–85.PubMedCrossRefGoogle Scholar
  29. 29.
    Songyang, Z. and Cantley, L.C., (1995) Recognition and specificity in protein tyrosine kinase-mediated signalling. Trends Biochem Sci, 20, 470–5.PubMedCrossRefGoogle Scholar
  30. 30.
    Songyang, Z. and Liu, D., (2001) Peptide library screening for determination of SH2 or phosphotyrosine-binding domain sequences. Methods Enzymol, 332, 183–95.PubMedCrossRefGoogle Scholar
  31. 31.
    Turk, B.E. and Cantley, L.C., (2003) Peptide libraries: at the crossroads of proteomics and bioinformatics. Curr Opin Chem Biol, 7, 84–90.PubMedCrossRefGoogle Scholar
  32. 32.
    Hutti, J.E., Jarrell, E.T., Chang, J.D., Abbott, D.W., Storz, P., Toker, A., et al., (2004) A rapid method for determining protein kinase phosphorylation specificity. Nat Methods, 1, 27–9.PubMedCrossRefGoogle Scholar
  33. 33.
    Sadowski, I., Stone, J.C. and Pawson, T., (1986) A noncatalytic domain conserved among cytoplasmic protein–tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps. Mol Cell Biol, 6, 4396–408.PubMedGoogle Scholar
  34. 34.
    Anderson, D., Koch, C.A., Grey, L., Ellis, C., Moran, M.F., and Pawson, T., (1990) Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. Science, 250, 979–82.PubMedCrossRefGoogle Scholar
  35. 35.
    Matsuda, M., Mayer, B.J., Fukui, Y., and Hanafusa, H., (1990) Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins. Science, 248, 1537–9.PubMedCrossRefGoogle Scholar
  36. 36.
    Songyang, Z., Shoelson, S.E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W.G., et al., (1993) SH2 domains recognize specific phosphopeptide sequences. Cell, 72, 767–78.PubMedCrossRefGoogle Scholar
  37. 37.
    Blaikie, P., Immanuel, D., Wu, J., Li, N., Yajnik, V., and Margolis, B., (1994) A region in Shc distinct from the SH2 domain can bind tyrosine-phosphorylated growth factor receptors. J Biol Chem, 269, 32031–4.PubMedGoogle Scholar
  38. 38.
    Kavanaugh, W.M. and Williams, L.T., (1994) An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science, 266, 1862–5.PubMedCrossRefGoogle Scholar
  39. 39.
    van der Geer, P., Wiley, S., Lai, V.K., Olivier, J.P., Gish, G.D., Stephens, R., et al., (1995) A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides. Curr Biol, 5, 404–12.PubMedCrossRefGoogle Scholar
  40. 40.
    Li, L., Wu, C., Huang, H., Zhang, K., Gan, J., and Li, S.S., (2008) Prediction of phosphotyrosine signaling networks using a scoring matrix-assisted ligand identification approach. Nucleic Acids Res, 36, 3263–73.Google Scholar
  41. 41.
    Pawson, T. and Nash, P., (2003) Assembly of cell regulatory systems through protein interaction domains. Science, 300, 445–52.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Shawn S.-C. Li
    • 1
  • Chenggang Wu
    • 1
  1. 1.Department of Biochemistry and the Siebens–Drake Medical Research Institute, Schulich School of Medicine and DentistryUniversity of Western OntarioLondonCanada

Personalised recommendations