Bioorthogonal Chemical Reporters for Analyzing Protein Sulfenylation in Cells

Thu H. Truong1, Kate S. Carroll2

1 Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 2 Department of Chemistry, The Scripps Research Institute, Jupiter, Florida
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110219
Online Posting Date:  June, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Protein sulfenylation (RSOH), the redox‐based modification of cysteine thiol side chains by hydrogen peroxide (H2O2), is an important mechanism in signal transduction. Likewise, dysregulated protein sulfenylation contributes to a range of human pathologies, including cancer. Efforts to elucidate the diverse roles of protein sulfenylation in physiology and disease have been hampered by the lack of techniques to probe these modifications in native environments. To address this problem, selective chemical reporters have been developed for the detection and identification of sulfenylated proteins directly in cells. In the approach described here, a cyclic β‐diketone warhead is functionalized with an azide or alkyne chemical handle. An orthogonally functionalized biotin or fluorescent reporter is then appended to the probe post‐homogenization via click chemistry for downstream analysis. These bi‐functional probes are exquisitely selective for protein sulfenyl modifications, non‐toxic, and do not perturb intracellular redox balance. These reagents have been utilized to investigate sulfenylation in vitro and to identify intracellular protein targets of H2O2 during cell signaling. These methods provide a facile way to detect protein sulfenic acids and to study the biological role of cysteine oxidation with regard to physiological and pathological events. Curr. Protoc. Chem. Biol. 4:101‐122 © 2012 by John Wiley & Sons, Inc.

Keywords: thiol modification; protein sulfenylation; redox signaling; click chemistry; in‐gel fluorescence; western blotting

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Labeling Sulfenylated Proteins In Vitro
  • Basic Protocol 2: Labeling Endogenous Sulfenylated Proteins in Cell Suspension
  • Basic Protocol 3: Labeling Exogenous Sulfenylated Proteins in Cell Suspension
  • Basic Protocol 4: Immunoblot Detection of Biotinylated Proteins
  • Basic Protocol 5: In‐Gel Detection of Fluorophore‐Tagged Proteins
  • Support Protocol 1: Pre‐Clearing Cell Lysates of Endogenous Biotinylated Proteins
  • Support Protocol 2: Methanol Precipitation of Proteins
  • Support Protocol 3: Methanol/Chloroform Precipitation of Proteins
  • Alternate Protocol 1: On‐Plate Labeling of Endogenous Protein Sulfenylation in Cells
  • Alternate Protocol 2: On‐Plate Labeling of Exogenous Protein Sulfenylation in Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Labeling Sulfenylated Proteins In Vitro

  Materials
  • GAPDH, lyophilized powder (Sigma‐Aldrich) or other purified protein of interest
  • Tris labeling buffer (see recipe)
  • 50 mM tris(2‐carboxyethyl) phosphine hydrochloride (TCEP; Sigma‐Aldrich), prepared fresh in water
  • Bio‐Spin 6 columns, pre‐packed in Tris buffer (BioRad)
  • DMSO (vehicle; Sigma‐Aldrich)
  • 25 mM DAz‐2 (Cayman Chemicals) or DYn‐2 (Cayman Chemicals), prepared in DMSO
  • 1 mM H 2O 2 stock (Sigma‐Aldrich), prepared fresh in water and maintained on ice
  • Click labeling buffer (see recipe)
  • 5 mM biotin tag (biotin alkyne or azide; Invitrogen) in DMSO or 5 mM fluorescent tag (TAMRA or AlexaFluor488 azide; Invitrogen) in DMSO
  • 2 mM tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl] amine (TBTA; Sigma Aldrich), prepared in 4:1 DMSO/t‐butanol (TBTA can also be synthesized by published methods; Chan et al., )
  • 50 mM CuSO 4, prepared fresh in water
  • 1× PBS (Boston BioProducts)
  • 2× Laemmli sample buffer with 10% β‐mercaptoethanol (BioRad)
  • Mini‐Protean TGX 4% to 15% Tris‐Glycine protein gels (BioRad)
  • Centrifuge
  • NanoDrop2000c spectrophotometer (Thermo Scientific)
  • 37°C incubator with shaker
  • Platform shaker
  • 95°C heating block

Basic Protocol 2: Labeling Endogenous Sulfenylated Proteins in Cell Suspension

  Materials
  • A431 cells (ATCC)
  • DMEM complete culture medium (high‐glucose DMEM supplemented with 10% FBS, 1% GlutaMax, 1% MEM nonessential amino acids, and 1% penicillin‐streptomycin; Invitrogen)
  • 1× PBS (Boston BioProducts)
  • DMEM only (serum‐free, high‐glucose DMEM; Invitrogen)
  • 30 µg/ml EGF stock (BD Biosciences), prepared in H 2O and kept on ice
  • 0.25% trypsin (Invitrogen)
  • 250 mM DAz‐2 (Cayman Chemicals) or DYn‐2 (Cayman Chemicals), prepared in DMSO
  • DMSO (vehicle; Sigma‐Aldrich)
  • Modified RIPA lysis buffer supplemented with EDTA‐free protease inhibitors and 200 U/ml catalase (see recipe)
  • BCA protein assay (Pierce)
  • 5 mM biotin tag (biotin alkyne or azide; Invitrogen) in DMSO or 5 mM fluorescent tag (TAMRA or AlexaFluor488 azide; Invitrogen) in DMSO
  • 50 mM TCEP (Sigma‐Aldrich), prepared fresh in water
  • 2 mM TBTA (Sigma Aldrich), prepared in 4:1 DMSO/t‐butanol (TBTA also synthesized by published methods; Chan et al., )
  • 50 mM CuSO 4, prepared fresh in water
  • 0.5 M EDTA (Boston BioProducts)
  • 10% SDS, prepared in H 2O
  • 2× Laemmli sample buffer with 10% β‐mercaptoethanol (BioRad)
  • Mini‐Protean TGX 4% to 15% Tris‐Glycine protein gels (BioRad)
  • 37°C incubator
  • Refrigerated centrifuge
  • 1.5‐ml microcentrifuge tubes
  • 95°C heating block
  • Vortex

Basic Protocol 3: Labeling Exogenous Sulfenylated Proteins in Cell Suspension

  Materials
  • HepG2 cells (ATCC)
  • MEM complete culture medium (MEM supplemented with 10% FBS, 1% GlutaMax, 1% MEM nonessential amino acids, and 1% penicillin‐streptomycin; Invitrogen)
  • 1× PBS (Boston BioProducts)
  • MEM with 0.5% FBS (MEM supplemented with 0.5% FBS; Invitrogen)
  • 100 mM H 2O 2 stock (Sigma‐Aldrich), prepared fresh in water and maintained on ice
  • Additional reagents and equipment (see protocol 2)

Basic Protocol 4: Immunoblot Detection of Biotinylated Proteins

  Materials
  • SDS‐PAGE gel with resolved samples
  • PVDF membrane (0.2‐µm; BioRad)
  • 3% BSA (Fisher), prepared in TBST
  • TBST (Boston BioProducts)
  • Streptavidin‐HRP antibody (GE‐Healthcare)
  • ECL Plus western blot detection system (GE Healthcare)
  • GAPDH antibody (Santa Cruz Biotechnology)
  • Rabbit anti‐mouse IgG‐HRP (Invitrogen)
  • X‐ray film

Basic Protocol 5: In‐Gel Detection of Fluorophore‐Tagged Proteins

  Materials
  • SDS‐PAGE gel with resolved samples
  • Destain solution (see recipe)
  • SYPRO ruby protein stain (BioRad)
  • Wash solution (see recipe)
  • Platform rocker
  • Fluorescence gel scanner (e.g., Amersham Biosciences Typhoon 9400 variable mode imager)

Support Protocol 1: Pre‐Clearing Cell Lysates of Endogenous Biotinylated Proteins

  Materials
  • NeutrAvidin agarose resin (Pierce)
  • Modified RIPA buffer (see recipe)
  • Cell lysates
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge
  • Platform rocker at 4°C

Support Protocol 2: Methanol Precipitation of Proteins

  Materials
  • Click chemistry reaction of cell lysate
  • Methanol, ice‐cold
  • Refrigerated centrifuge

Support Protocol 3: Methanol/Chloroform Precipitation of Proteins

  Materials
  • Click chemistry reaction of cell lysate
  • Methanol, ice‐cold
  • Chloroform, ice‐cold
  • Refrigerated centrifuge
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Benitez, L.V. and Allison, W.S. 1974. The inactivation of the acyl phosphatase activity catalyzed by the sulfenic acid form of glyceraldehyde 3‐phosphate dehydrogenase by dimedone and olefins. J. Biol. Chem. 249:6234‐6243.
   Chan, T.R., Hilgraf, R., Sharpless, K.B., and Fokin, V.V. 2004. Polytriazoles as copper(I)‐stabilizing ligands in catalysis. Org. Lett. 6:2853‐2855.
   Charron, G., Zhang, M.M., Yount, J.S., Wilson, J., Raghavan, A.S., Shamir, E., and Hang, H.C. 2009. Robust fluorescent detection of protein fatty‐acylation with chemical reporters. J. Am. Chem. Soc. 131:4967‐4975.
   Crane, E.J. 3rd, Vervoort, J., and Claiborne, A. 1997. 13C NMR analysis of the cysteine‐sulfenic acid redox center of enterococcal NADH peroxidase. Biochemistry 36:8611‐8618.
   Depuydt, M., Leonard, S.E., Vertommen, D., Denoncin, K., Morsomme, P., Wahni, K., Messens, J., Carroll, K.S., and Collet, J.F. 2009. A periplasmic reducing system protects single cysteine residues from oxidation. Science 326:1109‐1111.
   Dickinson, B.C. and Chang, C.J. 2011. Chemistry and biology of reactive oxygen species in signaling or stress responses. Nat. Chem. Biol. 7:504‐511.
   Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
   Lambeth, J.D. 2004. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4:181‐189.
   Leonard, S.E. and Carroll, K.S., 2011. Chemical omics' approaches for understanding protein cysteine oxidation in biology. Curr. Opin. Chem. Biol. 15:88‐102.
   Leonard, S.E., Reddie, K.G., and Carroll, K.S. 2009. Mining the thiol proteome for sulfenic acid modifications reveals new targets for oxidation in cells. ACS Chem. Biol. 4:783‐799.
   Leonard, S.E., Garcia, F.J., Goodsell, D.S., and Carroll, K.S. 2011. Redox‐based probes for protein tyrosine phosphatases. Angew. Chem. Int. Ed. Engl. 50:4423‐4427.
   Paulsen, C.E. and Carroll, K.S. 2009. Chemical dissection of an essential redox switch in yeast. Chem. Biol. 16:217‐225.
   Paulsen, C.E. and Carroll, K.S. 2010. Orchestrating redox signaling networks through regulatory cysteine switches. ACS Chem. Biol. 5:47‐62.
   Paulsen, C.E., Truong, T.H., Garcia, F.J., Homann, A., Gupta, V., Leonard, S.E., and Carroll, K.S. 2011. Peroxide‐dependent sulfenylation of the EGFR catalytic site enhances kinase activity. Nat. Chem. Biol. 8:57‐64.
   Poole, L.B., Klomsiri, C., Knaggs, S.A., Furdui, C.M., Nelson, K.J., Thomas, M.J., Fetrow, J.S., Daniel, L.W., and King, S.B. 2007. Fluorescent and affinity‐based tools to detect cysteine sulfenic acid formation in proteins. Bioconjug. Chem. 18:2004‐2017.
   Reddie, K.G. and Carroll, K.S. 2008. Expanding the functional diversity of proteins through cysteine oxidation. Curr. Opin. Chem. Biol. 12:746‐754.
   Reddie, K.G., Seo, Y.H., Muse, W.B. III, Leonard, S.E., and Carroll, K.S. 2008. A chemical approach for detecting sulfenic acid‐modified proteins in living cells. Mol. Biosyst. 4:521‐531.
   Rhee, S.G. 2006. Cell signaling. H2O2, a necessary evil for cell signaling. Science 312:1882‐1883.
   Rhee, S.G., Bae, Y.S., Lee, S.R., and Kwon, J. 2000. Hydrogen peroxide: A key messenger that modulates protein phosphorylation through cysteine oxidation. Sci. STKE 2000:pe1.
   Rostovtsev, V.V., Green, L.G., Fokin, V.V., and Sharpless, K.B. 2002. A stepwise huisgen cycloaddition process: Copper(I)‐catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41:2596‐2599.
   Salmeen, A., Andersen, J.N., Myers, M.P., Meng, T.C., Hinks, J.A., Tonks, N.K., and Barford, D. 2003. Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl‐amide intermediate. Nature 423:769‐773.
   Saxon, E. and Bertozzi, C.R. 2000. Cell surface engineering by a modified Staudinger reaction. Science 287:2007‐2010.
   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.
   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.
   Speers, A.E., Adam, G.C., and Cravatt, B.F. 2003. Activity‐based protein profiling in vivo using a copper(i)‐catalyzed azide‐alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125:4686‐4687.
   Truong, T.H., Garcia, F.J., Seo, Y.H., and Carroll, K.S. 2011. Isotope‐coded chemical reporter and acid‐cleavable affinity reagents for monitoring protein sulfenic acids. Bioorg. Med. Chem. Lett. 21:5015‐5020.
   Wilson, J.P., Raghavan, A.S., Yang, Y.Y., Charron, G., and Hang, H.C. 2011. Proteomic analysis of fatty‐acylated proteins in mammalian cells with chemical reporters reveals S‐acylation of histone H3 variants. Mol. Cell Proteomics 10:M110.001198.
   Winterbourn, C.C. 2008. Reconciling the chemistry and biology of reactive oxygen species. Nat. Chem. Biol. 4:278‐286.
   Wood, Z.A., Schroder, E., Robin Harris, J., and Poole, L.B. 2003. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28:32‐40.
   Yeh, J.I., Claiborne, A., and Hol, W.G. 1996. Structure of the native cysteine‐sulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8 Å resolution. Biochemistry 35:9951‐9957.
   Zheng, M., Aslund, F., and Storz, G. 1998. Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279:1718‐1721.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library