Detecting Protein Sulfenylation in Cells Exposed to a Toxicant

Phillip A. Wages1

1 Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.18
DOI:  10.1002/cptx.16
Online Posting Date:  February, 2017
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Abstract

Protein sulfenylation is a post‐translational modification that is linked to many cell signaling networks and specific protein functions, thus the detection of any sulfenylated protein after a toxicological exposure is of importance. Specifically, the detection of protein sulfenylation can provide multiple levels of mechanistic insight towards understanding the impact of a toxicological exposure. For instance, sulfenylation is caused by only a handful of reactive chemical species. Any altered sulfenylation suggests a change in cellular health, and the elucidation of the specific protein target that undergoes sulfenylation can help ascertain downstream targets and associated adverse outcomes. This document describes straightforward approaches to detect protein sulfenylation of total protein as well as individual proteins of interest with a focus on immunoblotting approaches. © 2017 by John Wiley & Sons, Inc.

Keywords: oxidative stress; sulfenylation; hydrogen peroxide; post‐translational modification; immunoblotting

     
 
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Table of Contents

  • Significance Statement
  • Introduction
  • Basic Protocol 1: Detection of Protein Sulfenylation in Cells
  • Basic Protocol 2: Avidin‐Blotting for Protein Sulfenylation Using Copper‐Catalyzed Alkyne‐Azide Cycloaddition
  • Alternate Protocol 1: Immunoprecipitaiton of Specific Sulfenylated Proteins Using Copper‐Catalyzed Alkyne‐Azide Cycloaddition
  • Support Protocol 1: Preclearing Lysates of Endogenous Biotin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Detection of Protein Sulfenylation in Cells

  Materials
  • Cell cultures in 100‐mm tissue culture dishes (BEAS‐2B epithelial cells)
  • Growth medium (KBM‐2 basal medium supplied with KGM‐2 Bullet Kit, Lonza)
  • Serum‐free cell culture medium (e.g., KBM‐2 basal medium, Lonza)
  • Phosphate‐buffered saline (PBS; ThermoFisher, cat. no. 10010023), 1×
  • 10 mM H 2O 2 in H 2O (deionized, distilled water)
  • 1 M Dimedone in dimethyl sulfoxide (DMSO)
  • 100 mM Dithiothreitol (DTT) in H 2O
  • Strong lysis buffer (see recipe), chilled on ice
  • 4× Laemmli sample buffer
  • Precast SDS‐PAGE gel
  • Tris‐buffered saline w/Tween 20 (TBST; see recipe)
  • Dry milk
  • Anti‐dimedone antibody (EMD Millipore, cat. no. 07‐2139 or EMD Millipore, cat. no. ABS30)
  • Secondary antibody against rabbit IgG
  • Cell culture incubator
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge, precooled to 4°C
  • Plate reader or spectrophotometer able to measure absorbance at 595 nm
  • Heating block capable of reaching 100°C
  • Nitrocellulose membrane

Basic Protocol 2: Avidin‐Blotting for Protein Sulfenylation Using Copper‐Catalyzed Alkyne‐Azide Cycloaddition

  Materials
  • Cell cultures in 100‐mm tissue culture dishes (BEAS‐2B Epithelial Cells)
  • Growth medium (KBM‐2 Basal medium supplied with the KGM‐2 Bullet Kit, Lonza)
  • Serum‐free cell culture medium (e.g., KBM‐2 Basal Medium, Lonza)
  • Phosphate‐buffered saline (PBS; ThermoFisher, cat. no. 10010023), 1×
  • 10 mM H 2O 2 in H 2O
  • 1 M DYn‐2 (Cayman Chemical, cat. no. 1354630‐46‐8) in dimethyl sulfoxide (DMSO)
  • 100 mM DTT in H 2O
  • Weak lysis buffer (see recipe), chilled on ice
  • Streptavidin beads, optional
  • 5 mM biotin‐azide in DMSO (ThermoFisher, cat. no. B10184)
  • 50 mM Tris(benzyltriazolymethyl)amine (TBTA) in dimethyl sulfoxide (DMSO)
  • 100 mM copper sulfate in H 2O
  • 100 mM sodium ascorbate in H 2O
  • 4× Laemmli sample buffer
  • Precast SDS‐PAGE gel
  • Biotin‐free blocking buffer (Pierce, cat. no. 37572)
  • Tris‐buffered saline with Tween 20 (TBST; see recipe)
  • Pierce High Sensitivity NeutrAvidin‐HRP (Pierce, cat. no. 31030)
  • ECL Western blotting substrate (Pierce, cat. no. 32106)
  • Cell culture incubator
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge, precooled to 4°C
  • Nitrocellulose membrane

Alternate Protocol 1: Immunoprecipitaiton of Specific Sulfenylated Proteins Using Copper‐Catalyzed Alkyne‐Azide Cycloaddition

  Additional Materials (also see protocol 2)
  • Protein A agarose
  • Primary antibody for protein of interest
  • Secondary antibody appropriate for primary antibody used
  • Wide‐orifice tips

Support Protocol 1: Preclearing Lysates of Endogenous Biotin

  Materials
  • Streptavidin‐conjugated resin (Thermo Scientific, cat. no. 29200)
  • Protein lysate
  • Weak lysis buffer (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge, precooled to 4°C
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Figures

Videos

Literature Cited

Literature Cited
  Barrett, T.J., Pattison, D.I., Leonard, S.E., Carroll, K.S., Davies, M.J., and Hawkins, C.L. 2012. Inactivation of thiol‐dependent enzymes by hypothiocyanous acid: Role of sulfenyl thiocyanate and sulfenic acid intermediates. Free Radic. Biol. Med. 52:1075‐1085. doi: 10.1016/j.freeradbiomed.2011.12.024.
  Burgoyne, J.R. and Eaton, P. 2011. Contemporary techniques for detecting and identifying proteins susceptible to reversible thiol oxidation. Biochem. Soc. Trans. 39:1260‐1267. doi: 10.1042/BST0391260.
  Chen, Y.X., Triola, G., and Waldmann, H. 2011. Bioorthogonal chemistry for site‐specific labeling and surface immobilization of proteins. Acc. Chem. Res. 44:762‐773. doi: 10.1021/ar200046h.
  Chen, X., Zhong, Z., Xu, Z., Chen, L., and Wang, Y. 2010. 2',7'‐Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy. Free Radic. Res. 44:587‐604. doi: 10.3109/10715761003709802.
  Cross, C.E., Valacchi, G., Schock, B., Wilson, M., Weber, S., Eiserich, J., and van der Vliet, A. 2002. Environmental oxidant pollutant effects on biologic systems: A focus on micronutrient antioxidant‐oxidant interactions. Am. J. Respir. Crit. Care Med. 166:S44‐50. doi: 10.1164/rccm.2206015.
  Crump, K.E., Juneau, D.G., Poole, L.B., Haas, K.M., and Grayson, J.M. 2012. The reversible formation of cysteine sulfenic acid promotes B‐cell activation and proliferation. Eur. J. Immunol. 42:2152‐2164. doi: 10.1002/eji.201142289.
  Hong, V., Steinmetz, N.F., Manchester, M., and Finn, M.G. 2010. Labeling live cells by copper‐ catalyzed alkyne–azide click chemistry. Bioconjug. Chem. 21:1912‐1916. doi: 10.1021/bc100272z.
  Janes, K.A. 2015. An analysis of critical factors for quantitative immunoblotting. Sci. Signal 8:rs2. doi: 10.1126/scisignal.2005966.
  Klomsiri, C., Karplus, P.A., and Poole, L.B. 2011. Cysteine‐based redox switches in enzymes. Antioxid. Redox Signal. 14:1065‐1077. doi: 10.1089/ars.2010.3376.
  Klomsiri, C., Nelson, K.J., Bechtold, E., Soito, L., Johnson, L.C., Lowther, W.T., Ryu, S.E., King, S.B., Furdui, C.M., and Poole, L.B. 2010. Use of dimedone‐based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox‐ sensitive proteins. Methods Enzymol. 473:77‐94. doi: 10.1016/S0076‐6879(10)73003‐2.
  Kulathu, Y., Garcia, F.J., Mevissen, T.E., Busch, M., Arnaudo, N., Carroll, K.S., Barford, D., and Komander, D. 2013. Regulation of A20 and other OTU deubiquitinases by reversible oxidation. Nat. Commun. 4:1569. doi: 10.1038/ncomms2567.
  Lee, S.R., Kwon, K.S., Kim, S.R., and Rhee, S.G. 1998. Reversible inactivation of protein‐ tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J. Biol. Chem. 273:15366‐15372. doi: 10.1074/jbc.273.25.15366.
  Maller, C., Schroder, E., and Eaton, P. 2011. Glyceraldehyde 3‐phosphate dehydrogenase is unlikely to mediate hydrogen peroxide signaling: Studies with a novel anti‐dimedone sulfenic acid antibody. Antioxid. Redox Signal. 14:49‐60. doi: 10.1089/ars.2010.3149.
  Martell, J. and Weerapana, E. 2014. Applications of copper‐catalyzed click chemistry in activity‐ based protein profiling. Molecules 19:1378‐1393. doi: 10.3390/molecules19021378.
  Nelson, K.J., Klomsiri, C., Codreanu, S.G., Soito, L., Liebler, D.C., Rogers, L.C., Daniel, L.W., and Poole, L.B. 2010. Use of dimedone‐based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins. Meth. Enzymol. 473:95‐115. doi: 10.1016/S0076‐6879(10)73004‐4.
  Paulsen, C.E., Truong, T.H., Garcia, F.J., Homann, A., Gupta, V., Leonard, S.E., and Carroll, K.S. 2012. Peroxide‐dependent sulfenylation of the EGFR catalytic site enhances kinase activity. Nat. Chem. Biol. 8:57‐64. doi: 10.1038/nchembio.736.
  Poole, L.B., Karplus, P.A., and Claiborne, A. 2004. Protein sulfenic acids in redox signaling. Annu. Rev. Pharmacol. Toxicol. 44:325‐347. doi: 10.1146/annurev.pharmtox.44.101802.121735.
  Seo, Y.H. and Carroll, K.S. 2009. Profiling protein thiol oxidation in tumor cells using sulfenic acid‐specific antibodies. Proc. Natl. Acad. Sci. U.S.A. 106:16163‐16168. doi: 10.1073/pnas.0903015106.
  Seo, Y.H. and Carroll, K.S. 2011. Quantification of protein sulfenic acid modifications using isotope‐coded dimedone and iododimedone. Angew. Chem. Int. Ed. Engl. 50:1342‐1345. doi: 10.1002/anie.201007175.
  Trujillo, M., Alvarez, B., and Radi, R. 2016. One‐ and two‐electron oxidation of thiols: Mechanisms, kinetics and biological fates. Free Radic. Res. 50:150‐171. doi: 10.3109/10715762.2015.1089988.
  Wages, P.A., Lavrich, K.S., Zhang, Z., Cheng, W.Y., Corteselli, E., Gold, A., Bromberg, P., Simmons, S.O., and Samet, J.M. 2015. Protein Sulfenylation: A Novel Readout of Environmental Oxidant Stress. Chem. Res. Toxicol. 28:2411‐2418. doi: 10.1021/acs.chemrestox.5b00424.
  Winterbourn, C.C. 2014. The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells. Biochim. Biophys. Acta 1840:730‐738. doi: 10.1016/j.bbagen.2013.05.004.
  Yang, J., Carroll, K.S., and Liebler, D.C. 2016. The Expanding Landscape of the Thiol Redox Proteome. Mol. Cell Proteomics 15:1‐11. doi: 10.1074/mcp.O115.056051.
  Yang, J., Gupta, V., Carroll, K.S., and Liebler, D.C. 2014. Site‐specific mapping and quantification of protein S‐sulfenylation in cells. Nat. Commun. 5:4776. doi: 10.1038/ncomms5776.
  Yang, J., Gupta, V., Tallman, K.A., Porter, N.A., Carroll, K.S., and Liebler, D.C. 2015. Global, in situ, site‐specific analysis of protein S‐sulfenylation. Nat. Protoc. 10:1022‐1037. doi: 10.1038/nprot.2015.062.
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