Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture


We have modified the biotin switch assay for protein S-nitrosothiols (SNOs), using resin-assisted capture (SNO-RAC). Compared with existing methodologies, SNO-RAC requires fewer steps, detects high-mass S-nitrosylated proteins more efficiently, and facilitates identification and quantification of S-nitrosylated sites by mass spectrometry. When combined with iTRAQ labeling, SNO-RAC revealed that intracellular proteins may undergo rapid denitrosylation on a global scale. This methodology is readily adapted to analyzing diverse cysteine-based protein modifications, including S-acylation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Analysis of protein S-nitrosylation by SNO-RAC.
Figure 2: iTRAQ-coupled SNO-RAC demonstrates proteome-wide (global) denitrosylation.

Similar content being viewed by others

Accession codes




  1. Hess, D.T. et al. Nat. Rev. Mol. Cell Biol. 6, 150–166 (2005).

    Article  CAS  Google Scholar 

  2. Cho, D.H. et al. Science 324, 102–105 (2009).

    Article  CAS  Google Scholar 

  3. Lim, K.H. et al. Nature 452, 646–649 (2008).

    Article  CAS  Google Scholar 

  4. Foster, M.W. et al. Trends Mol. Med. 9, 160–168 (2003).

    Article  CAS  Google Scholar 

  5. Jaffrey, S.R. et al. Nat. Cell Biol. 3, 193–197 (2001).

    Article  CAS  Google Scholar 

  6. Greco, T.M. et al. Proc. Natl. Acad. Sci. USA 103, 7420–7425 (2006).

    Article  CAS  Google Scholar 

  7. Paige, J.S. et al. Chem. Biol. 15, 1307–1316 (2008).

    Article  CAS  Google Scholar 

  8. Hao, G. et al. Proc. Natl. Acad. Sci. USA 103, 1012–1017 (2006).

    Article  CAS  Google Scholar 

  9. Roth, A.F. et al. Cell 125, 1003–1013 (2006).

    Article  CAS  Google Scholar 

  10. Hara, M.R. et al. Nat. Cell Biol. 7, 665–674 (2005).

    Article  CAS  Google Scholar 

  11. Forrester, M.T. et al. J. Biol. Chem. 282, 13977–13983 (2007).

    Article  CAS  Google Scholar 

  12. Benhar, M. et al. Science 320, 1050–1054 (2008).

    Article  CAS  Google Scholar 

  13. Liu, L. et al. Cell 116, 617–628 (2004).

    Article  CAS  Google Scholar 

  14. Ross, P.L. et al. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    Article  CAS  Google Scholar 

Download references


We thank S. Nimkar (Applied Biosystems) for providing iTRAQ reagents, and Q. Sun, K. Ozawa, A. Hausladen and D. Hess for expert advice. This work was supported by National Institutes of Health grants U19-ES012496, P01-HL075443, HL075443 and HL059130.

Author information

Authors and Affiliations



M.T.F., M.W.F. and L.N. performed experiments and analyzed data. J.W.T. and M.A.M. acquired and analyzed mass spectrometry data. M.T.F. and J.S.S. wrote the manuscript.

Corresponding author

Correspondence to Jonathan S Stamler.

Ethics declarations

Competing interests

Duke University (M.T.F., M.W.F., J.S.S.) has applied for a patent based on this methodology.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–3 (PDF 1291 kb)

Supplementary Table 4

MS spectra from all identified SNO-sites in CysNO-treated RAW264.7 macrophages and MG1655 E. coli. MSE (IdentityE) database search matches for the macrophage experiment are presented as Mascot searches for the purposes of display. (PDF 12223 kb)

Supplementary Table 5

Data from MS analyses of iTRAQ-coupled SNO-RAC. (ZIP 12668 kb)

Supplementary Table 6

MS/MS spectra from high mass protein-SNO identifications. (ZIP 1156 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Forrester, M., Thompson, J., Foster, M. et al. Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture. Nat Biotechnol 27, 557–559 (2009).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing