Analysis of Protein S‐Nitrosylation

Christopher M. Schonhoff1, Moran Benhar2

1 Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, 2 Technion‐Israel Institute of Technology, Haifa, Israel
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.6
DOI:  10.1002/0471140864.ps1406s63
Online Posting Date:  February, 2011
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Abstract

S‐Nitrosylation, the redox‐based modification of cysteine thiol side chains by nitric oxide, is a dynamic and reversible post‐translational modification of proteins that subserves many important cellular functions. Analysis of protein S‐nitrosylation is often challenging due to methodological limitations and the effects of various chemical and physical parameters. Despite these technical challenges, a growing number of useful methods are now available to analyze protein S‐nitrosylation. In this unit, several important methods to measure protein S‐nitrosylation and denitrosylation are discussed and evaluated. Recommendations are given regarding the potential and the applicability of the methods discussed. Curr. Protoc. Protein Sci. 63:14.6.1‐14.6.21. © 2011 by John Wiley & Sons, Inc.

Keywords: nitric oxide; S‐nitrosylation; Saville‐Griess assay; chemical reduction/chemiluminescence; DAN assay; biotin‐switch method

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: S‐Nitrosylation Analysis of Purified Recombinant Proteins Using a Saville‐Griess Assay
  • Basic Protocol 2: Analysis of Protein S‐Nitrosylation Using the Biotin‐Switch Method
  • Basic Protocol 3: S‐Nitrosylation Analysis of Purified Intracellular Proteins Using a DAN Assay
  • Basic Protocol 4: Analysis of Protein S‐Nitrosylation and Denitrosylation by Chemical Reduction/Chemiluminescence
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: S‐Nitrosylation Analysis of Purified Recombinant Proteins Using a Saville‐Griess Assay

  Materials
  • Purified recombinant protein of interest (Chapter 6)
  • HEN buffer (see recipe)
  • 1 mM S‐nitrosoglutathione (GSNO; see recipe) in HEN buffer (store up to 3 months in small aliquots at –70°C)
  • Solution A: 1% (w/v) sulfanilamide in 0.5 M HCl (store up to 1 month at room temperature protected from light)
  • Solution B: Solution A containing 0.2% (w/v) HgCl 2 (store up to 1 month at room temperature protected from light)
  • Solution C: 0.02% (w/v) N‐(1‐naphthyl)‐ethylenediamine dihydrochloride in 0.5 M HCl (store up to 1 month wrapped in foil at –20°C; if solution turns purple, discard)
  • Sephadex G‐25 column (PD‐10; GE Healthcare)
  • Spectrophotometer

Basic Protocol 2: Analysis of Protein S‐Nitrosylation Using the Biotin‐Switch Method

  Materials
  • Tissue culture cells of interest
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Cell lysis buffer (with protease inhibitors; see recipe)
  • HEN buffer (see recipe)
  • 25% (w/v) sodium dodecyl sulfate (SDS; appendix 2E) stock solution
  • 10% (v/v) S‐methyl methanethiosulfonate (MMTS; Fluka, cat. no. 64306) stock solution in N,N‐dimethylformamide (DMF)
  • Acetone, –20°C
  • 70% (v/v) acetone
  • HENS buffer: HEN buffer (see recipe) containing 1% (w/v) SDS [add 0.04 vol 25% (w/v) SDS]; stable up to 6 months at room temperature
  • 2.5 mg/ml N‐[6‐(biotinamido)hexyl]‐3′‐(2′‐pyridyldithio)propionamide (biotin‐HPDP; Pierce) stock solution in N,N‐dimethylformamide (DMF); prepare fresh for each experiment
  • 200 mM sodium ascorbate (Fluka), freshly prepared in HEN buffer (store in the dark on ice)
  • HENS/10 buffer: HEN/10 buffer (see recipe) with 1% (w/v) SDS
  • Neutralization buffer (see recipe)
  • 2× SDS‐PAGE sample buffer without reducing agents (see recipe)
  • 2.5% (w/v) nonfat dry milk (store up to 1 week at 4°C)
  • PBS ( appendix 2E) containing 0.1% (v/v) Tween 20 (store up to several years at 4°C)
  • Anti‐biotin mouse monoclonal primary antibody (Pierce)
  • Horseradish peroxidase–conjugated anti‐mouse secondary antibody (GE Healthcare)
  • Enhanced chemiluminescence (ECL) detection kit (GE Healthcare; also see unit 10.10)
  • Streptavidin agarose (Sigma) or NeutrAvidin agarose (Pierce)
  • HEN/10 buffer (HEN buffer diluted 10‐fold with H 2O)
  • Wash buffer: neutralization buffer (see recipe) containing 600 mM NaCl
  • Elution buffer (see recipe)
  • 2× SDS sample buffer with reducing agents (see recipe)
  • Antibodies specific for proteins of interest
  • Silver staining kit (optional; Sigma)
  • Refrigerated centrifuge (e.g., Sorvall, with RTH‐750 rotor)
  • Platform rocker
  • 50°C water bath
  • Additional reagents and equipment for protein assay (unit 3.4), SDS‐PAGE (unit 10.1), blotting onto nitrocellulose membranes (unit 10.7), enhanced chemiluminescence (ECL) detection (unit 10.10), and mass spectrometry (Chapter 16)

Basic Protocol 3: S‐Nitrosylation Analysis of Purified Intracellular Proteins Using a DAN Assay

  Materials
  • Cells expressing protein of interest
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • NP‐40 lysis buffer (see recipe) with and without freshly dissolved protease inhibitors, ice‐cold
  • 2 mg/ml protein A– or protein G–Sepharose bead slurry (GE Healthcare)
  • 0.4 µg/ml normal rabbit serum in NP‐40 lysis buffer
  • Antibody directed against the protein of interest
  • Isotype‐matched control antibody
  • High‐salt lysis buffer (see recipe), ice cold, containing freshly dissolved 1 mM N‐ethylmaleimide (NEM; add from 100 mM stock in ethanol) and protease inhibitors
  • PBS ( appendix 2E) containing 1 mM EDTA (store up to 1 year at 4°C)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Glycine elution buffer (see recipe)
  • Bovine serum albumin (BSA)
  • Silver staining kit (Sigma)
  • 10 mM 2,3‐diaminonapthalene (DAN) in dimethylformamide or dimethylsulfoxide (store up to 4 to 5 months at –20°C)
  • 100 mM HgCl 2 (store up to 1 year at room temperature)
  • 1 N NaOH
  • Refrigerated centrifuge (e.g., with Sorvall RTH‐750 rotor)
  • Platform rocker
  • Boiling water bath
  • Fluorimeter capable of excitation at 375 nm and emission detection at 450 nm
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)

Basic Protocol 4: Analysis of Protein S‐Nitrosylation and Denitrosylation by Chemical Reduction/Chemiluminescence

  Materials
  • Cells of interest
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Hypotonic lysis buffer (see recipe) with freshly dissolved protease inhibitors, ice cold.
  • HEN buffer (see recipe)
  • 100 mM CysNO stock solution (see recipe)
  • Reduced glutathione (GSH)
  • NADH
  • Acetone, –20°C
  • Helium or argon gas
  • 100 mM (100×) cysteine stock solution (prepare fresh)
  • Saturated (10 mM, 100×) CuCl solution (prepare fresh)
  • 1 mM S‐nitrosoglutathione (GSNO; see recipe) in HEN buffer (store up to 3 months in small aliquots at –70°C)
  • Refrigerated centrifuge
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Tube shaker
  • 10‐kDa MWCO centrifugal filters (Amicon)
  • Shaking water bath
  • Nitric oxide analyzer (NOA): Sievers NOA 280i (GE Analytical Instruments) or CLD 88 analyzer (Eco Medics, http://www.ecomedics.com/) and associated software
  • Hamilton syringe capable of delivering 100‐µl aliquot, airtight
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Figures

Videos

Literature Cited

Literature Cited
   Benhar, M., Forrester, M.T., Hess, D.T. and Stamler, J.S. 2008. Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins. Science 320:1050‐1054.
   Benhar, M., Forrester, M.T. and Stamler, J.S. 2009. Protein denitrosylation: Enzymatic mechanisms and cellular functions. Nat. Rev. 10:721‐732.
   Benhar, M., Thompson, J.W., Moseley, M.A. and Stamler, J.S. 2010. Identification of S‐nitrosylated targets of thioredoxin using a quantitative proteomic approach. Biochemistry 49:6963‐6969.
   Cook, J.A., Kim, S.Y., Teague, D., Krishna, M.C., Pacelli, R., Mitchell, J.B., Vodovotz, Y., Nims, R.W., Christodoulou, D., Miles, A.M., Grisham, M.B. and Wink, D.A. 1996. Convenient colorimetric and fluorometric assays for S‐nitrosothiols. Anal. Biochem. 238:150‐158.
   Fang, K., Ragsdale, N.V., Carey, R.M., Macdonald, T., and Gaston, B. 1998. Reductive assays for S‐nitrosothiols: Implications for measurements in biological systems. Biochem. Biophys. Res. Commun. 252:535‐540.
   Fernandez‐Cancio, M., Fernandez‐Vitos, E.M., Centelles, J.J., and Imperial, S. 2001. Sources of interference in the use of 2,3‐diaminonaphthalene for the fluorimetric determination of nitric oxide synthase activity in biological samples. Clin. Chim. Acta 312:205‐212.
   Forrester, M.T., Foster, M.W., and Stamler, J.S. 2007. Assessment and application of the biotin switch technique for examining protein S‐nitrosylation under conditions of pharmacologically induced oxidative stress. J. Biol. Chem. 282:13977‐13983.
   Forrester, M.T., Foster, M.W., Benhar, M., and Stamler, J.S. 2009a. Detection of protein S‐nitrosylation with the biotin‐switch technique. Free Radic. Biol. Med. 46:119‐126.
   Forrester, M.T., Thompson, J.W., Foster, M.W., Nogueira, L., Moseley, M.A., and Stamler, J.S. 2009b. Proteomic analysis of S‐nitrosylation and denitrosylation by resin‐assisted capture. Nat. Biotechnol. 27:557‐559.
   Foster, M.W., Hess, D.T., and Stamler, J.S. 2009. Protein S‐nitrosylation in health and disease: A current perspective. Trends Mol. Med. 15:391‐404.
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   Greco, T.M., Hodara, R., Parastatidis, I., Heijnen, H.F., Dennehy, M.K., Liebler, D.C., and Ischiropoulos, H. 2006. Identification of S‐nitrosylation motifs by site‐specific mapping of the S‐nitrosocysteine proteome in human vascular smooth muscle cells. Proc. Natl. Acad. Sci. U.S.A. 103:7420‐7425.
   Hao, G., Derakhshan, B., Shi, L., Campagne, F., and Gross, S.S. 2006. SNOSID, a proteomic method for identification of cysteine S‐nitrosylation sites in complex protein mixtures. Proc. Natl. Acad. Sci. U.S.A. 103:1012‐1017.
   Hara, M.R., Agrawal, N., Kim, S.F., Cascio, M.B., Fujimuro, M., Ozeki, Y., Takahashi, M., Cheah, J.H., Tankou, S.K., Hester, L.D., Ferris, C.D., Hayward, S.D., Snyder, S.H., and Sawa, A. 2005. S‐nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat. Cell Biol. 7:665‐674.
   Hess, D.T., Matsumoto, A., Kim, S.O., Marshall, H.E., and Stamler, J.S. 2005. Protein S‐nitrosylation: purview and parameters. Nat. Rev. 6:150‐166.
   Jaffrey, S.R., Erdjument‐Bromage, H., Ferris, C.D., Tempst, P., and Snyder, S.H. 2001. Protein S‐nitrosylation: A physiological signal for neuronal nitric oxide. Nat. Cell Biol. 3:193‐197.
   Liu, L., Hausladen, A., Zeng, M., Que, L., Heitman, J., and Stamler, J.S. 2001. A metabolic enzyme for S‐nitrosothiol conserved from bacteria to humans. Nature 410:490‐494.
   Mannick, J.B., Schonhoff, C., Papeta, N., Ghafourifar, P., Szibor, M., Fang, K., and Gaston, B. 2001. S‐Nitrosylation of mitochondrial caspases. J. Cell Biol. 154:1111‐1116.
   Nakamura, T. and Lipton, S.A. 2008. Emerging roles of S‐nitrosylation in protein misfolding and neurodegenerative diseases. Antioxid. Redox Signal. 10:87‐101.
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   Schonhoff, C.M., Matsuoka, M., Tummala, H., Johnson, M.A., Estevez, A.G., Wu, R., Kamaid, A., Ricart, K.C., Hashimoto, Y., Gaston, B., Macdonald, T.L., Xu, Z., and Mannick, J.B. 2006. S‐nitrosothiol depletion in amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U.S.A. 103:2404‐2409.
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