Chemical Proteomics pp 129-140

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

Identifying Cellular Targets of Small-Molecule Probes and Drugs with Biochemical Enrichment and SILAC

  • Shao-En Ong
  • Xiaoyu Li
  • Monica Schenone
  • Stuart L. Schreiber
  • Steven A. Carr


Sequencing of the human genome in the last decade has not yet led to a concomitant increase in the numbers of novel drug targets. While the pharmaceutical industry has invested heavily in improving drugs for existing protein targets, it has not tended toward a similar investment in experimental approaches to identify cellular targets of drugs. It is striking that the targets of numerous widely used FDA-approved drugs remain unknown. The development of robust, unbiased methods for target identification would greatly enhance our understanding the mechanisms-of-action of small molecules. Cell-based phenotypic screens followed by unbiased target identification have the potential to identify novel combinations of small molecules and their protein targets, shed light on drug polypharmacology, and enable unbiased screening approaches to drug discovery. Classical biochemical enrichment with immobilized small molecules has been used for over four decades but has been limited by issues concerning specificity and sensitivity. The application of mass spectrometry-based quantitative proteomics in combination with these affinity reagents has proven to be especially useful in addressing these common issues in affinity purification experiments. We describe the use of SILAC in identifying proteins that bind small-molecule probes and drugs in a cellular context.

Key words

Drug-target identification Proteomics SILAC Quantitation Small molecule Affinity chromatography 


  1. 1.
    Gamo, F. J., Sanz, L. M., Vidal, J., de Cozar, C., Alvarez, E., Lavandera, J. L., Vanderwall, D. E., Green, D. V., Kumar, V., Hasan, S., Brown, J. R., Peishoff, C. E., Cardon, L. R., and Garcia-Bustos, J. F. Thousands of chemical starting points for antimalarial lead identification, Nature 465, 305–310.Google Scholar
  2. 2.
    Guiguemde, W. A., Shelat, A. A., Bouck, D., Duffy, S., Crowther, G. J., Davis, P. H., Smithson, D. C., Connelly, M., Clark, J., Zhu, F., Jimenez-Diaz, M. B., Martinez, M. S., Wilson, E. B., Tripathi, A. K., Gut, J., Sharlow, E. R., Bathurst, I., El Mazouni, F., Fowble, J. W., Forquer, I., McGinley, P. L., Castro, S., Angulo-Barturen, I., Ferrer, S., Rosenthal, P. J., Derisi, J. L., Sullivan, D. J., Lazo, J. S., Roos, D. S., Riscoe, M. K., Phillips, M. A., Rathod, P. K., Van Voorhis, W. C., Avery, V. M., and Guy, R. K. Chemical genetics of Plasmodium falciparum, Nature 465, 311–315.Google Scholar
  3. 3.
    Stockwell, B. R. (2004) Exploring biology with small organic molecules, Nature 432, 846–854.PubMedCrossRefGoogle Scholar
  4. 4.
    Stanton, B. Z., Peng, L. F., Maloof, N., Nakai, K., Wang, X., Duffner, J. L., Taveras, K. M., Hyman, J. M., Lee, S. W., Koehler, A. N., Chen, J. K., Fox, J. L., Mandinova, A., and Schreiber, S. L. (2009) A small molecule that binds Hedgehog and blocks its signaling in human cells, Nat Chem Biol 5, 154–156.PubMedCrossRefGoogle Scholar
  5. 5.
    Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A. E., and Melton, D. A. (2008) Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds, Nat Biotechnol 26, 795–797.PubMedCrossRefGoogle Scholar
  6. 6.
    Ichida, J. K., Blanchard, J., Lam, K., Son, E. Y., Chung, J. E., Egli, D., Loh, K. M., Carter, A. C., Di Giorgio, F. P., Koszka, K., Huangfu, D., Akutsu, H., Liu, D. R., Rubin, L. L., and Eggan, K. (2009) A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog, Cell Stem Cell 5, 491–503.PubMedCrossRefGoogle Scholar
  7. 7.
    Rix, U., and Superti-Furga, G. (2009) Target profiling of small molecules by chemical proteomics, Nat Chem Biol 5, 616–624.PubMedCrossRefGoogle Scholar
  8. 8.
    Terstappen, G. C., Schlupen, C., Raggiaschi, R., and Gaviraghi, G. (2007) Target deconvolution strategies in drug discovery, Nat Rev Drug Discov 6, 891–903.PubMedCrossRefGoogle Scholar
  9. 9.
    Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and Mann, M. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Mol Cell Proteomics 1, 376–386.CrossRefGoogle Scholar
  10. 10.
    Ong, S. E., Schenone, M., Margolin, A. A., Li, X., Do, K., Doud, M. K., Mani, D. R., Kuai, L., Wang, X., Wood, J. L., Tolliday, N. J., Koehler, A. N., Marcaurelle, L. A., Golub, T. R., Gould, R. J., Schreiber, S. L., and Carr, S. A. (2009) Identifying the proteins to which small-molecule probes and drugs bind in cells, Proc Natl Acad Sci USA 106, 4617–4622.PubMedCrossRefGoogle Scholar
  11. 11.
    Ong, S. E., and Mann, M. (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC), Nat Protoc 1, 2650–2660.PubMedCrossRefGoogle Scholar
  12. 12.
    Ong, S. E., and Mann, M. (2005) Mass spectrometry-based proteomics turns quantitative, Nat Chem Biol 1, 252–262.PubMedCrossRefGoogle Scholar
  13. 13.
    Domon, B., and Aebersold, R. (2006) Mass spectrometry and protein analysis, Science 312, 212–217.PubMedCrossRefGoogle Scholar
  14. 14.
    Cox, J., and Mann, M. (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification, Nat Biotechnol 26, 1367–1372.PubMedCrossRefGoogle Scholar
  15. 15.
    Mortensen, P., Gouw, J. W., Olsen, J. V., Ong, S. E., Rigbolt, K. T., Bunkenborg, J., Cox, J., Foster, L., Heck, A. J., Blagoev, B., Andersen, J. S., and Mann, M. (2009) MSQuant, an open source platform for mass spectrometry-based quantitative proteomics, J Proteome Res. 9(1):393–403.CrossRefGoogle Scholar
  16. 16.
    Cuatrecasas, P. (1970) Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads, J Biol Chem 245, 3059–3065.Google Scholar
  17. 17.
    Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., and Mann, M. (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nat Protoc 1, 2856–2860.PubMedCrossRefGoogle Scholar
  18. 18.
    Rappsilber, J., Mann, M., and Ishihama, Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips, Nat Protoc 2, 1896–1906.PubMedCrossRefGoogle Scholar
  19. 19.
    Cox, J., Matic, I., Hilger, M., Nagaraj, N., Selbach, M., Olsen, J. V., and Mann, M. (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics, Nat Protoc 4, 698–705.PubMedCrossRefGoogle Scholar
  20. 20.
    Margolin, A. A., Ong, S. E., Schenone, M., Gould, R., Schreiber, S. L., Carr, S. A., and Golub, T. R. (2009) Empirical bayes analysis of quantitative proteomics experiments, PLoS One 4, e7454.PubMedCrossRefGoogle Scholar
  21. 21.
    Ting, L., Cowley, M. J., Hoon, S. L., Guilhaus, M., Raftery, M. J., and Cavicchioli, R. (2009) Normalization and statistical analysis of quantitative proteomics data generated by metabolic labeling, Mol Cell Proteomics 8, 2227–2242.PubMedCrossRefGoogle Scholar
  22. 22.
    Jiang, H., and English, A. M. (2002) Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine, J Proteome Res 1, 345–350.PubMedCrossRefGoogle Scholar
  23. 23.
    Kruger, M., Moser, M., Ussar, S., Thievessen, I., Luber, C. A., Forner, F., Schmidt, S., Zanivan, S., Fassler, R., and Mann, M. (2008) SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function, Cell 134, 353–364.PubMedCrossRefGoogle Scholar
  24. 24.
    Blagoev, B., Ong, S. E., Kratchmarova, I., and Mann, M. (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics, Nat Biotechnol 22, 1139–1145.PubMedCrossRefGoogle Scholar
  25. 25.
    Ong, S. E., Kratchmarova, I., and Mann, M. (2003) Properties of 13 C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC), J Proteome Res 2, 173–181.PubMedCrossRefGoogle Scholar
  26. 26.
    Bendall, S. C., Hughes, C., Stewart, M. H., Doble, B., Bhatia, M., and Lajoie, G. A. (2008) Prevention of amino acid conversion in SILAC experiments with embryonic stem cells, Mol Cell Proteomics 7, 1587–1597.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Shao-En Ong
    • 1
  • Xiaoyu Li
    • 2
  • Monica Schenone
    • 3
  • Stuart L. Schreiber
    • 4
  • Steven A. Carr
    • 3
  1. 1.Proteomics PlatformThe Broad Institute of MIT and HarvardCambridgeUSA
  2. 2.Chemical Biology platformThe Broad Institute of MIT and HarvardCambridgeUSA
  3. 3.Proteomics platformThe Broad Institute of MIT and HarvardCambridgeUSA
  4. 4.Chemical Biology Platform & Chemical Biology ProgramThe Broad Institute of MIT and HarvardCambridgeUSA

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