Methods to Detect Protein Glutathionylation

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 NaH₂ PO₄ , pH 7.4 ( appendix 2A )
100 mM GSSG (oxidized L‐glutathione) in H₂ O
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 H₂ O
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

Figure 6.17.1 Detection of global protein glutathionylation in GSSG‐treated BEAS‐2B cells by immunoblotting. BEAS‐2B cells in serum‐free DMEM/F12 medium were treated with 10 mM GSSG for 1 hr. Whole‐cell extracts were prepared using NP‐40 cell lysis buffer containing 20 mM NEM. For DTT‐treated samples, extracts were treated with 10 mM DTT for 10 min at room temperature. Cell extracts (20 µg) were resolved by nonreducing SDS‐PAGE, and global protein glutathionylation and GAPDH expression were analyzed by immunoblotting. The presence of anti‐GSH immunoreactive bands indicates cellular proteins that are potentially glutathionylated. Glutathionylation is readily reversible under reducing conditions, and the loss of the anti‐GSH immunoreactive bands in the DTT‐treated samples confirms which bands represent glutathionylated proteins. In this case, all the anti‐GSH immunoreactive bands except one at ∼160 kDa represent glutathionylated protein, as this band is present in the DTT‐treated sample. Equivalent protein loading amounts are demonstrated by the similar relative densities of the anti‐GAPDH immunoreactive bands. These results demonstrate that BEAS‐2B cells have a low level of basal protein glutathionylation that is dramatically increased by treatment with GSSG.

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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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Methods to Detect Protein Glutathionylation

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited