Advertisement

Affinity-Based Chemoproteomics with Small Molecule-Peptide Conjugates

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 803)

Abstract

In affinity-based chemoproteomics strategies, the direct immobilization of small bioactive probe molecules to a solid support may pose problems with respect to the preservation of the functional activity toward the target proteins. Typically, immobilized molecules on solid supports exhibit lower affinity for target proteins compared to the free parent molecule. This may lead to a failure to specifically capture the target proteins or to unacceptable losses during the washing steps. To circumvent these shortcomings, we have devised small molecule-peptide conjugates (SMPCs), which enable wide-ranging experimental strategies for the capturing of protein targets of small molecules from cells or tissues. With the possibilities of synthesizing peptides of tailored biochemical and biophysical properties, SMPCs enable the identification of protein targets of small molecules from cell-lysates and intact cells. Moreover, labeling of these conjugates with fluorophores can provide information on the cellular localization and distribution of the target.

Key words

Peptide-coupled small molecule Immuno-chemoproteomics Small molecule-peptide conjugates Drug target deconvolution Target protein capture from live cells Target identification 

References

  1. 1.
    Saxena, C., Higgs, R.E., Zhen, E., and Hale, J.E. (2009) Small-molecule affinity chromatography coupled mass spectrometry for drug target deconvolution. Expert Opinion on Drug Discovery 4, 701–714.CrossRefGoogle Scholar
  2. 2.
    Hahn, R., Berger, E., Pflegerl, K., and Jungbauer, A. (2003) Directed immobilization of peptide ligands to accessible pore sites by conjugation with a placeholder molecule. Anal. Chem. 75, 543–8.PubMedCrossRefGoogle Scholar
  3. 3.
    Saxena, C., Zhen, E., Higgs, R.E., and Hale, J.E. (2008) An Immuno-Chemo-Proteomics Method for Drug Target Deconvolution. J Proteome Res. 7, 3490–3497.PubMedCrossRefGoogle Scholar
  4. 4.
    Saxena C., Bonacci, T.M., Huss, K.L., Bloem, L.J., Higgs, R.E., and Hale, J.E. (2009) Capture of Drug Targets from Live Cells Using a Multipurpose Immuno-Chemo-Proteomics Tool. J Proteome Res. 8, 3951–3957.PubMedCrossRefGoogle Scholar
  5. 5.
    Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., and Loriolle, F. (1991) The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 15771–15781.PubMedGoogle Scholar
  6. 6.
    Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Shantani Proteome Analytics Pvt. Ltd.PuneIndia

Personalised recommendations