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

Nature Biotechnology volume 27, pages 557–559 (2009)Cite this article

Abstract

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.

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

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA

    Michael T Forrester & Jonathan S Stamler

  2. Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA

    Michael T Forrester

  3. Proteomics Core Facility, Duke University Medical Center, Durham, North Carolina, USA

    J Will Thompson & M Arthur Moseley

  4. Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA

    Matthew W Foster, Leonardo Nogueira & Jonathan S Stamler

Authors
  1. Michael T Forrester
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Contributions

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.

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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)

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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). https://doi.org/10.1038/nbt.1545

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