Methods to Detect Protein Glutathionylation

Robyn L. Poerschke1, Kristofer S. Fritz1, Christopher C. Franklin2

1 Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, Anschutz Medical Campus, University of Colorado Denver, Aurora, Colorado, 2 University of Colorado Cancer Center, Anschutz Medical Campus, University of Colorado Denver, Aurora, Colorado
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.17
DOI:  10.1002/0471140856.tx0617s57
Online Posting Date:  September, 2013
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Abstract

Glutathionylation is a posttranslational modification that results in the formation of a mixed disulfide between glutathione and the thiol group of a protein cysteine residue. Glutathionylation of proteins occurs via both nonenzymatic mechanisms involving thiol/disulfide exchange and enzyme‐mediated reactions. Protein glutathionylation is observed in response to oxidative or nitrosative stress and is redox‐dependent, being readily reversible under reducing conditions. Such findings suggest that glutathionylation plays an important role in mediating redox‐sensitive signaling. Indeed, glutathionylation can affect protein function by altering activity, protein‐protein interactions, and ligand binding. Glutathionylation may also serve to prevent cysteine residues from undergoing irreversible oxidative modification. Thus, determining the ability of a given protein to become glutathionylated can provide insight into its redox regulation and putative role in dictating cellular response to oxidative and nitrosative stress. Methods to measure protein glutathionylation using immunoblotting and mass spectrometry are described. Curr. Protoc. Toxicol. 57:6.17.1‐6.17.18. © 2013 by John Wiley & Sons, Inc.

Keywords: glutathionylation; glutathione; posttranslational modification; immunoblotting; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Glutathionylation of Recombinant Protein and Detection by Immunoblotting
  • Basic Protocol 2: Detection of Protein Glutathionylation by Mass Spectrometry
  • Basic Protocol 3: Determination of Global Cellular Protein Glutathionylation by Immunoblotting
  • Alternate Protocol 1: Detecting the Glutathionylation of a Specific Cellular Protein by GSH Immunoprecipitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Glutathionylation of Recombinant Protein and Detection by Immunoblotting

  Materials
  • 8 µM recombinant protein for protein of interest
  • 50 mM NaH 2PO 4, pH 7.4 ( appendix 2A)
  • 100 mM GSSG (oxidized L‐glutathione) in H 2O
  • 100 mM DTT ( appendix 2A)
  • 4× nonreducing SDS‐PAGE loading buffer (see recipe)
  • 10% Bis‐Tris acrylamide mini gel (or appropriate percentage gel for the molecular weight of the protein of interest)
  • Protein standards for SDS‐PAGE
  • Tris‐glycine SDS running buffer (see recipe)
  • TBS‐T (see recipe)
  • 100% methanol
  • Immunoblotting transfer buffer (see recipe)
  • TBS‐T containing 5% (w/v) nonfat dry milk
  • Anti‐GSH mouse monoclonal primary antibody (ViroGen), 1:100 dilution in TBS‐T containing 0.5% (w/v) nonfat dry milk
  • Horseradish peroxidase (HRP)‐conjugated secondary antibody against mouse IgG, 1:5000 dilution in TBS‐T
  • Enhanced chemiluminescence (ECL) reagent
  • Stripping buffer (see recipe)
  • Primary antibody against protein of interest and corresponding species‐specific HRP‐conjugated secondary antibody, 1:1000 dilution in TBS‐T
  • Mini‐PROTEAN 3 Electrophoresis System with Mini‐Trans‐Blot electrophoretic transfer cell (Bio‐Rad)
  • 0.45‐µm PVDF Immobilon transfer membrane
  • Immunoblotting filter paper
  • Hybridization oven prewarmed to 50°C

Basic Protocol 2: Detection of Protein Glutathionylation by Mass Spectrometry

  Materials
  • Polyacrylamide gel containing GSSG‐treated recombinant protein separated by nonreducing SDS‐PAGE (see protocol 1)
  • Coomassie brilliant blue R‐250 staining solution (see recipe)
  • Destaining solution (see recipe)
  • 50 mM ammonium bicarbonate, pH 8.0
  • 50% acetonitrile in 50 mM ammonium bicarbonate
  • 100% acetonitrile
  • 0.1 µg/µl trypsin
  • 5% formic acid in acetonitrile
  • 0.1% trifluoroacetic acid (TFA) in water
  • 0.1% TFA in 60% acetonitrile
  • α‐cyano‐4‐hydroxycinnamic acid (CHCA) MALDI matrix
  • Glassware washed in 10% acetic acid for preparing solutions, staining, and destaining
  • (Optional) Flat‐tipped curved forceps (e.g., Wiha #44521) for handling gel fragments
  • 37°C water bath or temperature‐controlled oven
  • Sonicating water bath
  • C18 ZipTips (Millipore)
  • ABI SCIEX 4800 Plus MALDI‐TOF/TOF mass spectrometer with Opti‐TOF 96 well insert
  • ProteoMass Calibration Kit (Sigma‐Aldrich)

Basic Protocol 3: Determination of Global Cellular Protein Glutathionylation by Immunoblotting

  Materials
  • Cell cultures in 100‐mm tissue culture dishes
  • Serum‐free cell culture media
  • 100 mM GSSG (oxidized L‐glutathione) in H 2O
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • Cell lysis buffer (see recipe), ice cold
  • Bicinchoninic acid (BCA) protein assay kit
  • 1 M DTT
  • 4× nonreducing SDS loading buffer
  • Anti‐GAPDH rabbit polyclonal antibody (or primary antibody against another loading control protein), 1:1000 dilution in TBS‐T containing 0.5% (w/v) nonfat dry milk
  • TBS‐T (see recipe)
  • HRP‐conjugated secondary antibody against rabbit IgG, 1:5000 dilution in TBS‐T
  • Sonicator
  • Microcentrifuge, precooled to 4°C
  • Clear, flat‐bottom 96‐well plate
  • Microplate reader capable of measuring A 562
  • PD‐10 protein desalting spin columns
  • Additional reagents and equipment for SDS‐PAGE, immunoblotting, detection, and visualization ( protocol 1)

Alternate Protocol 1: Detecting the Glutathionylation of a Specific Cellular Protein by GSH Immunoprecipitation

  Materials
  • Whole‐cell extract ( protocol 3)
  • Cell lysis buffer (see recipe)
  • Anti‐GSH mouse monoclonal primary antibody (ViroGen)
  • Protein A/G agarose beads
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • 1× nonreducing SDS loading buffer (see recipe)
  • Primary antibody for protein of interest and corresponding species‐specific HRP‐conjugated secondary antibody
  • Microcentrifuge tube rotator in 4°C refrigerator or cold room
  • Additional reagents and equipment for SDS‐PAGE, immunoblotting, detection, and visualization ( protocol 1)
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Figures

Videos

Literature Cited

  Backos, D.S., Fritz, K.S., Roede, J.R., Petersen, D.R., and Franklin, C.C. 2011. Posttranslational modification and regulation of glutamate‐cysteine ligase by the alpha,beta‐unsaturated aldehyde 4‐hydroxy‐2‐nonenal. Free Radic. Biol. Med. 50:14‐26.
  Brennan, J.P., Miller, J.I., Fuller, W., Wait, R., Begum, S., Dunn, M.J., and Eaton, P. 2006. The utility of N,N‐biotinyl glutathione disulfide in the study of protein S‐glutathiolation. Mol. Cell. Proteomics 5:215‐225.
  Dave, K.A., Headlam, M.J., Wallis, T.P., and Gorman, J.J. 2011. Preparation and analysis of proteins and peptides using MALDI TOF/TOF mass spectrometry. Curr. Protoc. Protein Sci. 63:16.13.1‐16.13.21.
  Findlay, V.J., Townsend, D.M., Morris, T.E., Fraser, J.P., He, L., and Tew, K.D. 2006. A novel role for human sulfiredoxin in the reversal of glutathionylation. Cancer Res. 66:6800‐6806.
  Fritz, K.S., Kellersberger, K.A., Gomez, J.D., and Petersen, D.R. 2012. 4‐HNE adduct stability characterized by collision‐induced dissociation and electron transfer dissociation mass spectrometry. Chem. Res. Toxicol. 25:965‐970.
  Hill, B.G. and Bhatnagar, A. 2012. Protein S‐glutathiolation: Redox‐sensitive regulation of protein function. J. Mol. Cell. Cardiol. 52:559‐567.
  Liao, S., Ewing, N.P., Boucher, B., Materne, O., and Brummel, C.L. 2012. High‐throughput screening for glutathione conjugates using stable‐isotope labeling and negative electrospray ionization precursor‐ion mass spectrometry. Rapid Commun. Mass Spectrom. 26:659‐669.
  Menon, D. and Board, P.G. 2013. A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3‐naphthalenedicarboxaldehyde. Anal. Biochem. 433:132‐136.
  Niture, S.K., Velu, C.S., Bailey, N.I., and Srivenugopal, K.S. 2005. S‐thiolation mimicry: Quantitative and kinetic analysis of redox status of protein cysteines by glutathione‐affinity chromatography. Arch. Biochem. Biophys. 444:174‐184.
  Priora, R., Coppo, L., Salzano, S., Di Simplicio, P., and Ghezzi, P. 2010. Measurement of mixed disulfides including glutathionylated proteins. Meth. Enzymol. 473:149‐159.
  Shelton, M.D., Chock, P.B., and Mieyal, J.J. 2005. Glutaredoxin: Role in reversible protein S‐glutathionylation and regulation of redox signal transduction and protein translocation. Antioxid. Redox. Signal. 7:348‐366.
  Townsend, D.M., Manevich, Y., He, L., Hutchens, S., Pazoles, C.J., and Tew, K.D. 2009. Novel role for glutathione S‐transferase pi. Regulator of protein S‐glutathionylation following oxidative and nitrosative stress. J. Biol. Chem. 284:436‐445.
  Wu, S.L., Jiang, H., Hancock, W.S., and Karger, B.L. 2010. Identification of the unpaired cysteine status and complete mapping of the 17 disulfides of recombinant tissue plasminogen activator using LC‐MS with electron transfer dissociation/collision induced dissociation. Anal. Chem. 82:5296‐5303.
  Xiong, Y., Uys, J.D., Tew, K.D., and Townsend, D.M. 2011. S‐glutathionylation: From molecular mechanisms to health outcomes. Antioxid. Redox. Signal. 15:233‐270.
Internet Resources
  http://www.abcam.com/ps/pdf/protocols/WB‐beginner.pdf
  Web site provides a guide to immunoblotting, with sections on sample preparation, electrophoresis parameters, transfer conditions, and blot visualization. It includes modifications to utilize when immunoblotting large proteins (>100 kDa).
  http://www.bio‐rad.com/LifeScience/pdf/Bulletin_2895.pdf
  Additional guide to immunoblotting, with emphasis on transfer and detection methods. This guide includes a troubleshooting section.
  http://www.matrixscience.com
  Web site for the Mascot database search engine used to analyze mass spectrometry data. The site includes tutorials for database searching.
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