Drug Affinity Responsive Target Stability (DARTS) to Resolve Protein–Small Molecule Interaction in Arabidopsis

Cecilia Rodriguez‐Furlan1, Chunhua Zhang2, Natasha Raikhel1, Glenn R. Hicks1

1 Center for Plant Cell Biology, Institute for Integrative Genome Biology, and Department of Botany and Plant Sciences, University of California, Riverside, California, 2 Purdue Center for Plant Biology, Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20062
Online Posting Date:  December, 2017
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Target identification remains a challenging step in plant chemical genomics approaches. Drug affinity responsive target stability (DARTS) represents a straightforward technique to identify small molecules’ protein targets and assist in the characterization of interactions between small molecules and putative targets identified by other methods. When a small molecule interacts with a protein, it has the potential to stabilize the protein's structure, resulting in a reduced susceptibility to protease action. During the DARTS procedure, protein extracts are treated with proteolytic enzymes, and only proteins that bind to the small molecule are protected from proteolysis. DARTS represents a protocol independent of the molecule's mechanism of action or chemical structure. Another advantage of DARTS is that it does not require additional modifications or tagging of the small molecule. The protocols outlined in this article describe in detail the DARTS technique applied to plant proteins and propose several detection procedures according to protein abundance. © 2017 by John Wiley & Sons, Inc.

Keywords: Arabidopsis; DARTS; Pronase; small molecule; target identification

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: DARTS Using Arabidopsis Complex Protein Lysate
  • Support Protocol 1: Immunodetection by Western Blot
  • Support Protocol 2: Detection by Gel Staining and Mass Spectrometry
  • Support Protocol 3: Detection by Gel‐Free Mass Spectrometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: DARTS Using Arabidopsis Complex Protein Lysate

  • Arabidopsis tissue
  • Liquid nitrogen
  • Protein extraction buffer (see recipe), 4°C
  • 5 mg/ml small molecule of interest in DMSO
  • Dimethylsulfoxide (DMSO)
  • 10 mg/ml Pronase enzyme solution in water
  • 20× protease inhibitor solution (see recipe)
  • 6× Laemmli buffer (see recipe)
  • Mortar and pestle or automatic grinder
  • Spoon, precooled
  • 50‐ml conical tubes (e.g., Corning Falcon), precooled
  • Miracloth
  • Refrigerated centrifuge
  • 1.5‐ml microcentrifuge tubes
  • Mechanical rotator
  • Hotplate incubator
  • Centrifuge tube cap clips or cap holders
  • Microcentrifuge
  • Additional reagents and equipment for SDS‐PAGE (unit 7.3; Gallagher, ), western blotting (unit 8.3; Ni, Xu, Sabanayagam, & Gallagher, ), protein staining (unit 7.4; Gallagher & Sasse, ), MS proteomics (see protocol 3 and unit 16.4; Wither, Hansen, & Reisz, ), and tandem MS (see protocol 4 and unit 23.1; Link & Washburn, )

Support Protocol 1: Immunodetection by Western Blot

  Additional Materials (also see protocol 1Basic Protocol)
  • Trans‐illuminator
  • Clean scalpel or razor blade
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Literature Cited

Literature Cited
  Aniento, F., & Gruenberg, J. (2003). Subcellular fractionation of tissue culture cells. Current Protocols in Protein Science, 32, 4.3.1–4.3.21. doi: 10.1002/0471140864.ps0403s32.
  Dejonghe, W., & Russinova, E. (2014). Target identification strategies in plant chemical biology. Frontiers in Plant Science, 5, 352. doi: 10.3389/fpls.2014.00352.
  Drakakaki, G., Robert, S., Szatmari, A.‐M., Brown, M. Q., Nagawa, S., Van Damme, D. … Hicks, G. R. (2011). Clusters of bioactive compounds target dynamic endomembrane networks in vivo. Proceedings of the National Academy of Sciences, 108(43), 17850–17855. doi: 10.1073/pnas.1108581108.
  Gallagher, S. R. (2012). SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). Current Protocols Essential Laboratory Techniques, 6, 7.3.1–7.3.28. doi: 10.1002/9780470089941.et0703s06.
  Gallagher, S. R., & Sasse, J. (2012). Staining proteins in gels. Current Protocols Essential Laboratory Techniques, 6, 7.4.1–7.4.14. doi: 10.1002/9780470089941.et0704s06.
  Halder, V., & Kombrink, E. (2015). Facile high‐throughput forward chemical genetic screening by in situ monitoring of glucuronidase‐based reporter gene expression in Arabidopsis thaliana. Frontiers in Plant Science, 6, 13. doi: 0.3389/fpls.2015.00013.
  Hicks, G. R., & Raikhel, N. V. (2012). Small molecules present large opportunities in plant biology. Annual Review of Plant Biology, 63, 261–282. doi: 10.1146/annurev‐arplant‐042811‐105456.
  Kapp, E., & Schütz, F. (2007). Overview of tandem mass spectrometry (MS/MS) database search algorithms. Current Protocols in Protein Science, 49, 25.2.1–25.2.19. doi: 10.1002/0471140864.ps2502s49.
  Knoth, C., Salus, M. S., Girke, T., & Eulgem, T. (2009). The synthetic elicitor 3,5‐dichloroanthranilic acid induces NPR1‐dependent and NPR1‐independent mechanisms of disease resistance in Arabidopsis. Plant Physiology, 150(1), 333–347. doi: 10.1104/pp.108.133678.
  LaMontagne, E. D., Collins, C. A., Peck, S. C., & Heese, A. (2016). Isolation of microsomal membrane proteins from Arabidopsis thaliana. Current Protocols in Plant Biology, 1, 217–234. doi: 10.1002/cppb.20020.
  Li, R., Rodriguez Furlan, C., Wang, J., van de Ven, W., Gao, T., Raikhel, N. V., & Hicks, G. R. (2016). Different endomembrane trafficking pathways establish apical and basal polarities. The Plant Cell, 29(1), 90–108. 10.1105/tpc.16.00524.
  Link, A. J., & Washburn, M. P. (2014). Analysis of protein composition using multidimensional chromatography and mass spectrometry. Current Protocols in Protein Science, 78, 23.1.1–23.1.25. doi: 10.1002/0471140864.ps2301s78.
  Lomenick, B., Hao, R., Jonai, N., Chin, R. M., Aghajan, M., Warburton, S.… Huang, J. (2009). Target identification using drug affinity responsive target stability (DARTS). Proceedings of the National Academy of Sciences, 106(51), 21984–21989. doi: 10.1073/pnas.0910040106.
  Ni, D., Xu, P., Sabanayagam, D., & Gallagher, S. R. (2016). Protein blotting: Immunoblotting. Current Protocols Essential Laboratory Techniques, 12, 8.3.1–8.3.40. doi: 10.1002/9780470089941.et0803s12.
   Noutoshi, Y., Ikeda, M., Saito, T., Osada, H., & Shirasu, K. (2012). Sulfonamides identified as plant immune‐priming compounds in high‐throughput chemical screening increase disease resistance in Arabidopsis thaliana. Frontiers in Plant Science, 3, 245. doi: 10.3389/fpls.2012.00245.
  Rodriguez‐Furlan, C., Hicks, G. R., & Norambuena, L. (2014). Chemical genomics: characterizing target pathways for bioactive compounds using the endomembrane trafficking network. Methods in Molecular Biology, 1174, 317–28. doi: 10.1007/978‐1‐4939‐0944‐5_22.
  Wither, M. J., Hansen, K. C., & Reisz, J. A. (2016). Mass spectrometry‐based bottom‐up proteomics: Sample preparation, LC‐MS/MS analysis, and database query strategies. Current Protocols in Protein Science, 86, 16.4.1–16.4.20. doi: 10.1002/cpps.18.
  Zhang, C., Brown, M. Q., van de Ven, W., Zhang, Z.‐M., Wu, B., Young, M. C.…Pan, S. (2016). Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis. Proceedings of the National Academy of Sciences, 113(1), E41–E50. doi: 10.1073/pnas.1521248112.
  Zhang, G., Annan, R. S., Carr, S. A., & Neubert, T. A. (2014). Overview of peptide and protein analysis by mass spectrometry. Current Protocols in Molecular Biology, 108, 10.21.1–10.21.30. doi: 10.1002/0471142727.mb1021s108.
  Zhao, Y., Chow, T. F., Puckrin, R. S., Alfred, S. E., Korir, A. K., Larive, C. K., & Cutler, S. R. (2007). Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated. Nature Chemical Biology, 3(11), 716–721. doi: 10.1038/nchembio.2007.32.
  Zumstein, L. (1995). Dialysis. Current Protocols in Protein Science, 00, A.3B.1–A.3B.4. doi: 10.1002/0471140864.psa03bs00.
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