Target Identification Using Drug Affinity Responsive Target Stability (DARTS)

Brett Lomenick1, Gwanghyun Jung1, James A. Wohlschlegel2, Jing Huang2

1 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, 2 Molecular Biology Institute, University of California Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110180
Online Posting Date:  December, 2011
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Abstract

Drug affinity responsive target stability (DARTS) is a general methodology for identifying and studying protein‐ligand interactions. The technique is based on the principle that when a small molecule compound binds to a protein, the interaction stabilizes the target protein's structure such that it becomes resistant to proteases. DARTS is particularly useful for the initial identification of the protein targets of small molecules, but can also be used to validate potential protein‐ligand interactions predicted or identified by other means and to estimate the affinity of interactions. The approach is simple and advantageous because it can be performed using crude cell lysates and other complex protein mixtures (without requiring purified proteins), and it uses native, unmodified small molecules. The protocols in this unit describe the general approach for performing DARTS experiments, which can be easily modified and scaled to fit project‐specific criteria. Curr. Protoc. Chem. Biol. 3:163‐180 © 2011 by John Wiley & Sons, Inc.

Keywords: target ID; DARTS; proteins; ligands; binding; proteomics; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: DARTS Experiment for Analyzing Drug Binding to Protein Targets in Mammalian Cell Lysates with Pronase
  • Basic Protocol 2: Thermolysin Digestion DARTS Experiment for Analyzing Binding of Small Molecules to Protein Targets in Yeast Cell Lysates
  • Support Protocol 1: Preparation of DARTS Samples for MudPIT Analysis
  • Support Protocol 2: Analysis of DARTS Samples by In‐Solution Molecular Weight–Based Fractionation and Mass Spectrometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: DARTS Experiment for Analyzing Drug Binding to Protein Targets in Mammalian Cell Lysates with Pronase

  Materials
  • M‐PER lysis buffer (Pierce, cat. no. 78501)
  • Phosphatase inhibitor solutions (100 mM β‐pyrophosphate, 50 mM sodium pyrophosphate, 200 mM sodium vanadate, and 1 M sodium fluoride)
  • 20× Protease inhibitor solution (see )
  • Phosphate‐buffered saline [PBS; 1× PBS was diluted from 10× PBS (MPbio, cat. no. PBS10X02) using filter‐sterilized distilled water]
  • 10‐cm dish cell culture in logarithmic growth phase (up to 70% to 80% confluent)
  • 10× TNC buffer (see recipe)
  • BCA protein concentration assay (Pierce, cat. no. 23225 or similar assay)
  • Dimethyl sulfoxide (DMSO; or other corresponding vehicle)
  • 100× stock solution of small molecule(s) in DMSO (DMSO can be replaced with other vehicle of choice)
  • Pronase stock solution (see recipe)
  • 1× TNC buffer (dilute 10× TNC buffer 10‐fold in dH 2O)
  • Cell scraper (e.g., Fisher, cat. no. 08‐100‐241)
  • 1.5‐ml tubes
  • Refrigerated centrifuge capable of 18,000 × g (e.g., Beckman Microcentrifuge 22R)

Basic Protocol 2: Thermolysin Digestion DARTS Experiment for Analyzing Binding of Small Molecules to Protein Targets in Yeast Cell Lysates

  Materials
  • 2× Triton X‐100 lysis buffer (see recipe)
  • 4× Phosphatase inhibitor solution (see recipe)
  • 20× Protease inhibitor solution: (see recipe)
  • Wild‐type strain of S. cerevisiae grown to mid‐log phase (∼2 × 107 cells/ml)
  • BCA protein concentration assay (Pierce, cat. no. 23225 or similar assay)
  • 1× TNC Buffer (diluted from 10× TNC buffer; see recipe for 10× TNC buffer)
  • Dimethyl sulfoxide (DMSO)
  • 100× small molecule stock solution in DMSO
  • Thermolysin stock solution (see recipe)
  • 0.5 M EDTA (pH 8.0)
  • Refrigerated centrifuge capable of 18,000 × g (e.g., Beckman microcentrifuge 22R)
  • 1.5‐ml tubes
  • 0.5‐mm glass beads (e.g., BioSpec Products, cat. no. 11079105)
  • Vortex mixer
  • 21‐G needles
  • Benchtop microcentrifuge

Support Protocol 1: Preparation of DARTS Samples for MudPIT Analysis

  • TE buffer (see recipe)
  • Protein samples (see protocol 1 or protocol 2)
  • 100% (w/v) trichloroacetic acid (TCA; Sigma, cat. no. T0699)
  • HPLC‐grade acetone (Fisher, cat. no. AC26831)
  • 8 M urea, 100 mM Tris⋅Cl, pH 8.5 (prepared fresh)
  • 200 mM Tris(2‐carboxylethyl)‐phosphine hydrochloride (TCEP)
  • 500 mM Iodoacetamide (prepared fresh)
  • Sequencing‐grade endoproteinase Lys‐C (Princeton Separations, cat. no. EN‐130)
  • 100 mM Tris, pH 8.5
  • 100 mM CaCl 2
  • Sequencing‐grade trypsin
  • 90% formic acid (HPLC grade; e.g.,Fisher, cat. no. PI‐28905 diluted with HPLC‐grade water to 90% final concentration)
  • Vivacon 500 10K MWCO Spin Columns (Sartorius Stedim, cat. no. VN01H01)

Support Protocol 2: Analysis of DARTS Samples by In‐Solution Molecular Weight–Based Fractionation and Mass Spectrometry

  • 1 M dithiothreitol (DTT)
  • 5× sample buffer (provided with the cartridge kit)
  • Zeba Spin Desalting Columns, 7K MWCO, 0.5 ml (Thermo Scientific, cat. no. 89882)
  • 10% Tris‐acetate cartridge kit (Protein Discovery, cat. no. 42105)
  • Gelfree 8100 Fractionation Station (Protein Discovery)
  • Additional reagents and equipment for filter‐aided sample preparation (FASP; Wisniewski et al., )
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Figures

Videos

Literature Cited

   Aghajan, M., Jonai, N., Flick, K., Fu, F., Luo, M., Cai, X., Ouni, I., Pierce, N., Tang, X., Lomenick, B., Damoiseaux, R., Hao, R., Del Moral, P.M., Verma, R., Li, Y., Li, C., Houk, K.N., Jung, M.E., Zheng, N., Huang, L., Deshaies, R.J., Kaiser, P., and Huang, J. 2010. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nat. Biotechnol. 28:738‐742.
   Arnold, U., Rucknagel, K.P., Schierhorn, A., and Ulbrich‐Hofmann, R. 1996. Thermal unfolding and proteolytic susceptibility of ribonuclease A. Eur. J. Biochem. 237:862‐869.
   Chen, T., Ozel, D., Qiao, Y., Harbinski, F., Chen, L., Denoyelle, S., He, X., Zvereva, N., Supko, J.G., Chorev, M., Halperin, J.A., and Aktas, B.H. 2011. Chemical genetics identify eIF2alpha kinase heme‐regulated inhibitor as an anticancer target. Nat. Chem. Biol. 7:610‐616.
   Ghaemmaghami, S., Huh, W.K., Bower, K., Howson, R.W., Belle, A., Dephoure, N., O'Shea, E.K., and Weissman, J.S. 2003. Global analysis of protein expression in yeast. Nature 425:737‐741.
   Gordon, J. 1991. Use of vanadate as protein‐phosphotyrosine phosphatase inhibitor. Methods Enzymol. 201:477‐482.
   Lomenick, B., Hao, R., Jonai, N., Chin, R.M., Aghajan, M., Warburton, S., Wang, J., Wu, R.P., Gomez, F., Loo, J.A., Wohlschlegel, J.A., Vondriska, T.M., Pelletier, J., Herschman, H.R., Clardy, J., Clarke, C.F., and Huang, J. 2009. Target identification using drug affinity responsive target stability (DARTS). Proc. Natl. Acad. Sci. U.S.A. 106:21984‐21989.
   Lomenick, B., Olsen, R.W., and Huang, J. 2011. Identification of direct protein targets of small molecules. ACS Chem. Biol. 6:34‐46.
   Martzen, M.R., McCraith, S.M., Spinelli, S.L., Torres, F.M., Fields, S., Grayhack, E.J. and Phizicky, E.M. 1999. A biochemical genomics approach for identifying genes by the activity of their products. Science 286:1153‐1155.
   Ross‐Macdonald, P., Coelho, P.S., Roemer, T., Agarwal, S., Kumar, A., Jansen, R., Cheung, K.H., Sheehan, A., Symoniatis, D., Umansky, L., Heidtman, M., Nelson, F.K., Iwasaki, H., Hager, K., Gerstein, M., Miller, P., Roeder, G.S., and Snyder, M. 1999. Large‐scale analysis of the yeast genome by transposon tagging and gene disruption. Nature 402:413‐418.
   Washburn, M.P. 2008. Sample preparation and in‐solution protease digestion of proteins for chromatography‐based proteomic analysis. Curr. Protoc. Prot. Sci. 53:23.6.1‐23.6.11.
   Washburn, M.P., Wolters, D., and Yates, J.R. 3rd. 2001. Large‐scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19:242‐247.
   Wisniewski, J.R., Zougman, A., Nagaraj, N., and Mann, M. 2009. Universal sample preparation method for proteome analysis. Nat. Methods 6:359‐362.
   Wolters, D.A., Washburn, M.P., and Yates, J.R. 3rd. 2001. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73:5683‐5690.
   Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R.A., Gerstein, M., and Snyder, M. 2001. Global analysis of protein activities using proteome chips. Science 293:2101‐2105.
Internet Resources
   http://labs.pharmacology.ucla.edu/huanglab/DARTS_faq.html
  DARTS Help Forum. This provides real‐time updated answers and discussions for the frequently asked questions from the community.
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