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Identification of S-Nitrosothiols by the Sequential Cysteine Blocking Technique

  • Rafael A. Homem
  • Thierry Le Bihan
  • Manda Yu
  • Gary J. Loake
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1424)

Abstract

Here, we describe a procedure for the identification of S-nitrosothiols that has been used in our laboratory to study the roles of protein S-nitrosylation in the immune responses of Arabidopsis thaliana and other organisms. It employs a modified version of the biotin-switch technique, which we termed the sequential cysteine blocking technique, encompassing the sequential redox-blocking of recombinant proteins followed by LC–MS/MS analysis.

Key words

S-nitrosylation S-nitrosothiols Redox-based posttranslational modification LC–MS/MS Iodoacetamide Sequential cysteine blocking Label-free quantitation 

References

  1. 1.
    Yu M, Lamattina L, Spoel S, Loake G (2014) Nitric oxide function in plant biology: a redox cue in deconvolution. New Phytol 202(4):1142–1156CrossRefPubMedGoogle Scholar
  2. 2.
    Tsang A, Lee Y, Ko H (2009) S-nitrosylation of XIAP compromises neuronal survival in Parkinson’s disease. Proc Natl Acad Sci 106(12):4900–4905CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Spoel S, Tada Y, Loake G (2010) Post-translational protein modification as a tool for transcription reprogramming. New Phytol 186(2):333–339CrossRefPubMedGoogle Scholar
  4. 4.
    Sha Y, Marshall HE (2012) S-nitrosylation in the regulation of gene transcription. Biochim Biophys Acta 1820(6):701–711CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Nakamura T, Tu S, Akhtar M, Sunico C (2013) Aberrant protein s-nitrosylation in neurodegenerative diseases. Neuron 78(4):596–614CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Feechan A, Kwon E, Yun B-W, Wang Y, Pallas JA, Loake GJ (2005) A central role for S-nitrosothiols in plant disease resistance. Proc Natl Acad Sci U S A 102(22):8054–8059CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wang Y-Q, Feechan A, Yun B-W, Shafiei R, Hofmann A, Taylor P, Xue P, Yang F-Q, Xie Z-S, Pallas JA, Chu C-C, Loake GJ (2009) S-nitrosylation of AtSABP3 antagonizes the expression of plant immunity. J Biol Chem 284(4):2131–2137CrossRefPubMedGoogle Scholar
  8. 8.
    Tada Y, Spoel SH, Pajerowska-Mukhtar K, Mou Z, Song J, Wang C, Zuo J, Dong X (2008) Plant immunity requires conformational changes [corrected] of NPR1 via S-nitrosylation and thioredoxins. Science 321(5891):952–956CrossRefPubMedGoogle Scholar
  9. 9.
    Yun B-W, Feechan A, Yin M, Saidi NBB, Le Bihan T, Yu M, Moore JW, Kang J-G, Kwon E, Spoel SH, Pallas JA, Loake GJ (2011) S-nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature 478(7368):264–268CrossRefPubMedGoogle Scholar
  10. 10.
    Astier J, Besson-Bard A, Lamotte O, Bertoldo J, Bourque S, Terenzi H, Wendehenne D (2012) Nitric oxide inhibits the ATPase activity of the chaperone-like AAA+ ATPase CDC48, a target for S-nitrosylation in cryptogein signalling in tobacco cells. Biochem J 447(2):249–260CrossRefPubMedGoogle Scholar
  11. 11.
    Loake G, Grant M (2007) Salicylic acid in plant defence--the players and protagonists. Curr Opin Plant Biol 10(5):466–472CrossRefPubMedGoogle Scholar
  12. 12.
    Slaymaker DH, Navarre DA, Clark D, del Pozo O, Martin GB, Klessig DF (2002) The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. Proc Natl Acad Sci U S A 99(18):11640–11645CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kumar D, Klessig DF (2003) High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and has salicylic acid-stimulated lipase activity. Proc Natl Acad Sci U S A 100(26):16101–16106CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Hoang CV, Chapman KD (2002) Biochemical and molecular inhibition of plastidial carbonic anhydrase reduces the incorporation of acetate into lipids in cotton embryos and tobacco cell suspensions and leaves. Plant Physiol 128(4):1417–1427CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kachroo P, Shanklin J, Shah J, Whittle EJ, Klessig DF (2001) A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Proc Natl Acad Sci 98(16):9448–9453CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kwon E, Feechan A, Yun B-W, Hwang B-H, Pallas JA, Kang J-G, Loake GJ (2012) AtGSNOR1 function is required for multiple developmental programs in Arabidopsis. Planta 236(3):887–900CrossRefPubMedGoogle Scholar
  17. 17.
    Wang P, Du Y, Hou Y-J, Zhao Y, Hsu C-C, Yuan F, Zhu X, Tao WA, Song C-P, Zhu J-K (2015) Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1. Proc Natl Acad Sci U S A 112(2):613–618CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Feng J, Wang C, Chen Q, Chen H, Ren B, Li X, Zuo J (2013) S-nitrosylation of phosphotransfer proteins represses cytokinin signaling. Nat Commun 4:1529CrossRefPubMedGoogle Scholar
  19. 19.
    Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH (2001) Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol 3(2):193–197CrossRefPubMedGoogle Scholar
  20. 20.
    Camerini S, Polci M, Bachi A (2005) Proteomics approaches to study the redox state of cysteine-containing proteins. Ann Ist Super Sanita 41(4):451–457PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Rafael A. Homem
    • 1
  • Thierry Le Bihan
    • 2
  • Manda Yu
    • 1
  • Gary J. Loake
    • 1
    • 2
  1. 1.Institute of Molecular Plant Sciences, School of Biological SciencesUniversity of EdinburghEdinburghUK
  2. 2.Synthetic and Systems Biology, School of Biological SciencesUniversity of EdinburghEdinburghUK

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