Nitric Oxide pp 253-266 | Cite as

Biotin Switch Processing and Mass Spectrometry Analysis of S-Nitrosated Thioredoxin and Its Transnitrosation Targets

  • Changgong Wu
  • Tong Liu
  • Yan Wang
  • Lin Yan
  • Chuanlong Cui
  • Annie Beuve
  • Hong LiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)


S-Nitrosation is a key posttranslational modification in regulating proteins in both normal physiology and diverse human diseases. To identify novel therapies for human diseases linked to oxidative and nitrosative stress, understanding how cells control S-nitrosation specificity could be critical. Among the enzymes known to control S-nitrosation of proteins, thioredoxin 1 (Trx1), a conserved disulfide reductase, transnitrosates and denitrosates distinct sets of target proteins. To recognize the function of Trx1 in both normal and dysfunctional cells, S-nitrosation targets of Trx1 in different cells need to be identified. However, S-nitrosation is usually too labile to be detected directly by mass spectrometry (MS). Here we present two optimized MS techniques to identify S-nitrosated Trx1 and its transnitrosation targets, using both direct and indirect MS methods.

Key words

S-nitrosation Thioredoxin Transnitrosation Mass spectrometry 



This chapter is produced with a grant support from the NIH-National Institute of General Medical Sciences (R01GM112415, to A.B. and H.L.), and the Orbitrap MS described here was purchased with a grant from the NIH-National Institute of Neurological Disorders and Stroke (P30NS046593).


  1. 1.
    Foster MW, Hess DT, Stamler JS (2009) Protein S-nitrosylation in health and disease: a current perspective. Trends Mol Med 15(9):391–404CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Mannick JB (2007) Regulation of apoptosis by protein S-nitrosylation. Amino Acids 32(4):523–526CrossRefPubMedGoogle Scholar
  3. 3.
    Benhar M, Forrester MT, Hess DT, Stamler JS (2008) Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins. Science 320(5879):1050–1054CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Gao C, Guo H, Wei J, Mi Z, Wai PY, Kuo PC (2005) Identification of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages. Nitric Oxide 12(2):121–126CrossRefPubMedGoogle Scholar
  5. 5.
    Wu C, Liu T, Chen W, Oka S, Fu C, Jain MR, Parrott AM, Baykal AT, Sadoshima J, Li H (2010) Redox regulatory mechanism of transnitrosylation by thioredoxin. Mol Cell Proteomics 9(10):2262–2275Google Scholar
  6. 6.
    Haendeler J, Hoffmann J, Tischler V, Berk BC, Zeiher AM, Dimmeler S (2002) Redox regulatory and anti-apoptotic functions of thioredoxin depend on S-nitrosylation at cysteine 69. Nat Cell Biol 4(10):743–749CrossRefPubMedGoogle Scholar
  7. 7.
    Haendeler J, Weiland U, Zeiher AM, Dimmeler S (1997) Effects of redox-related congeners of NO on apoptosis and caspase-3 activity. Nitric Oxide 1(4):282–293CrossRefPubMedGoogle Scholar
  8. 8.
    Hashemy SI, Holmgren A (2008) Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues. J Biol Chem 283(32):21890–21898Google Scholar
  9. 9.
    Wu C, Parrott AM, Fu C, Liu T, Marino SM, Gladyshev VN, Jain MR, Baykal AT, Li Q, Oka S, Sadoshima J, Beuve A, Simmons WJ, Li H (2011) Thioredoxin 1-mediated post-translational modifications: reduction, transnitrosylation, denitrosylation, and related proteomics methodologies. Antioxid Redox Signal 15(9):2565–2604Google Scholar
  10. 10.
    Greco TM, Hodara R, Parastatidis I, Heijnen HF, Dennehy MK, Liebler DC, 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(19):7420–7425Google Scholar
  11. 11.
    Hao G, Derakhshan B, Shi L, Campagne F, Gross SS (2006) SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures. Proc Natl Acad Sci U S A 103(4):1012–1017Google Scholar
  12. 12.
    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
  13. 13.
    Paige JS, Xu G, Stancevic B, Jaffrey SR (2008) Nitrosothiol reactivity profiling identifies S-nitrosylated proteins with unexpected stability. Chem Biol 15(12):1307–1316CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Torta F, Usuelli V, Malgaroli A, Bachi A (2008) Proteomic analysis of protein S-nitrosylation. Proteomics 8(21):4484–4494CrossRefPubMedGoogle Scholar
  15. 15.
    Gow AJ, Davis CW, Munson D, Ischiropoulos H (2004) Immunohistochemical detection of S-nitrosylated proteins. Methods Mol Biol 279:167–172PubMedGoogle Scholar
  16. 16.
    Jaffrey SR, Snyder SH (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001(86):PL1PubMedGoogle Scholar
  17. 17.
    Huang B, Chen C (2006) An ascorbate-dependent artifact that interferes with the interpretation of the biotin switch assay. Free Radic Biol Med 41(4):562–567CrossRefPubMedGoogle Scholar
  18. 18.
    Knipp M, Braun O, Gehrig PM, Sack R, Vasák M (2003) Zn(II)-free dimethylargininase-1 (DDAH-1) is inhibited upon specific Cys-S-nitrosylation. J Biol Chem 278(5):3410–3416CrossRefPubMedGoogle Scholar
  19. 19.
    Wang Y, Liu T, Wu C, Li H (2008) A strategy for direct identification of protein S-nitrosylation sites by quadrupole time-of-flight mass spectrometry. J Am Soc Mass Spectrom 19(9):1353–1360CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Lee SJ, Lee JR, Kim YH, Park YS, Park SI, Park HS, Kim KP (2007) Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 21(17):2797–2804CrossRefPubMedGoogle Scholar
  21. 21.
    Wu C, Parrott AM, Liu T, Beuve A, Li H (2013) Functional proteomics approaches for the identification of transnitrosylase and denitrosylase targets. Methods 62(2):151–160Google Scholar
  22. 22.
    Wu C, Parrott AM, Liu T, Jain MR, Yang Y, Sadoshima J, Li H (2011) Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach. J Proteome 74(11):2498–2509Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Changgong Wu
    • 1
  • Tong Liu
    • 1
  • Yan Wang
    • 2
  • Lin Yan
    • 1
  • Chuanlong Cui
    • 1
  • Annie Beuve
    • 3
  • Hong Li
    • 1
    Email author
  1. 1.Center for Advanced Proteomics Research and Department of Microbiology, Biochemistry and Molecular GeneticsRutgers, New Jersey Medical SchoolNewarkUSA
  2. 2.School of PharmacyShanghai Jiao Tong UniversityShanghaiP. R. China
  3. 3.Department of Pharmacology, Physiology and NeuroscienceRutgers, New Jersey Medical SchoolNewarkUSA

Personalised recommendations