Skip to main content
SpringerLink
Log in

Detection of Disulfide Linkage by Chemical Derivatization and Mass Spectrometry

  • Protocol
  • First Online:
Polyadenylation in Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1255))

Abstract

The location of disulfide linkage(s) or status of unpaired cysteines is a critical structural feature required for the characterization of three-dimensional structure of a protein and for the correlation of protein structure–function relationships. Cysteine, with its reactive thiol group, can undergo enzymatic or oxidative posttranslational modification in response to changing redox conditions to signal a cascade of downstream reactions. In such a situation, it becomes even more critical to obtain the information on the pair of cysteines involved in such a redox switch operation. Here, a method involving chemical derivatization and liquid chromatography–mass spectrometry (LC-MS) is described to determine the cysteine residues involved in disulfide bond formation for a protein containing multiple cysteines in its sequence.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gilmartin GM (2005) Eukaryotic mRNA 3′ processing: a common means to different ends. Genes Dev 19(21):2517–2521. doi:10.1101/gad.1378105

    Article  CAS  PubMed  Google Scholar 

  2. Hunt AG, Xing D, Li QQ (2012) Plant polyadenylation factors: conservation and variety in the polyadenylation complex in plants. BMC Genomics 13:641. doi:10.1186/1471-2164-13-641

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Hunt AG, Xu R, Addepalli B, Rao S, Forbes KP, Meeks LR, Xing D, Mo M, Zhao H, Bandyopadhyay A, Dampanaboina L, Marion A, Von Lanken C, Li QQ (2008) Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling. BMC Genomics 9:220. doi:10.1186/1471-2164-9-220

    Article  PubMed Central  PubMed  Google Scholar 

  4. Mandel CR, Bai Y, Tong L (2008) Protein factors in pre-mRNA 3′-end processing. Cell Mol Life Sci 65(7–8):1099–1122. doi:10.1007/s00018-007-7474-3

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Zhang J, Addepalli B, Yun KY, Hunt AG, Xu R, Rao S, Li QQ, Falcone DL (2008) A polyadenylation factor subunit implicated in regulating oxidative signaling in Arabidopsis thaliana. PLoS One 3(6):e2410. doi:10.1371/journal.pone.0002410

    Article  PubMed Central  PubMed  Google Scholar 

  6. Buchanan BB, Balmer Y (2005) Redox regulation: a broadening horizon. Annu Rev Plant Biol 56:187–220. doi:10.1146/annurev.arplant.56.032604.144246

    Article  CAS  PubMed  Google Scholar 

  7. Bykova NV, Rampitsch C (2013) Modulating protein function through reversible oxidation: redox-mediated processes in plants revealed through proteomics. Proteomics 13(3–4):579–596. doi:10.1002/pmic.201200270

    Article  CAS  PubMed  Google Scholar 

  8. Paulsen CE, Carroll KS (2010) Orchestrating redox signaling networks through regulatory cysteine switches. ACS Chem Biol 5(1):47–62. doi:10.1021/cb900258z

    Article  CAS  PubMed  Google Scholar 

  9. Winterbourn CC (2008) Reconciling the chemistry and biology of reactive oxygen species. Nat Chem Biol 4(5):278–286. doi:10.1038/nchembio.85

    Article  CAS  PubMed  Google Scholar 

  10. Murray CI, Van Eyk JE (2012) Chasing cysteine oxidative modifications: proteomic tools for characterizing cysteine redox status. Circ Cardiovasc Genet 5(5):591. doi:10.1161/CIRCGENETICS.111.961425

    Article  PubMed Central  PubMed  Google Scholar 

  11. Bachi A, Dalle-Donne I, Scaloni A (2013) Redox proteomics: chemical principles, methodological approaches and biological/biomedical promises. Chem Rev 113(1):596–698. doi:10.1021/cr300073p

    Article  CAS  PubMed  Google Scholar 

  12. Chouchani ET, James AM, Fearnley IM, Lilley KS, Murphy MP (2011) Proteomic approaches to the characterization of protein thiol modification. Curr Opin Chem Biol 15(1):120–128. doi:10.1016/j.cbpa.2010.11.003

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Han B, Hare M, Wickramasekara S, Fang Y, Maier CS (2012) A comparative ‘bottom up’ proteomics strategy for the site-specific identification and quantification of protein modifications by electrophilic lipids. J Proteomics 75(18):5724–5733. doi:10.1016/j.jprot.2012.07.029

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Izquierdo-Alvarez A, Martinez-Ruiz A (2011) Thiol redox proteomics seen with fluorescent eyes: the detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE. J Proteomics 75(2):329–338. doi:10.1016/j.jprot.2011.09.013

    Article  CAS  PubMed  Google Scholar 

  15. Gallegos-Perez JL, Rangel-Ordonez L, Bowman SR, Ngowe CO, Watson JT (2005) Study of primary amines for nucleophilic cleavage of cyanylated cystinyl proteins in disulfide mass mapping methodology. Anal Biochem 346(2):311–319. doi:10.1016/j.ab.2005.08.003

    Article  CAS  PubMed  Google Scholar 

  16. Wefing S, Schnaible V, Hoffmann D (2006) SearchXLinks. A program for the identification of disulfide bonds in proteins from mass spectra. Anal Chem 78(4):1235–1241. doi:10.1021/ac051634x

    Article  CAS  PubMed  Google Scholar 

  17. Bach RD, Dmitrenko O, Thorpe C (2008) Mechanism of thiolate-disulfide interchange reactions in biochemistry. J Org Chem 73(1):12–21. doi:10.1021/jo702051f

    Article  CAS  PubMed  Google Scholar 

  18. Addepalli B, Hunt AG (2008) Ribonuclease activity is a common property of Arabidopsis CCCH-containing zinc-finger proteins. FEBS Lett 582(17):2577–2582. doi:10.1016/j.febslet.2008.06.029

    Article  CAS  PubMed  Google Scholar 

  19. Addepalli B, Limbach PA, Hunt AG (2010) A disulfide linkage in a CCCH zinc finger motif of an Arabidopsis CPSF30 ortholog. FEBS Lett 584(21):4408–4412. doi:10.1016/j.febslet.2010.09.043

    Article  CAS  PubMed  Google Scholar 

  20. Eng JK, McCormack AL, Yates Iii JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5(11):976–989. doi:10.1016/1044-0305(94)80016-2

    Article  CAS  PubMed  Google Scholar 

  21. Cottrell JS (2011) Protein identification using MS/MS data. J Proteomics 74(10):1842–1851. doi:10.1016/j.jprot.2011.05.014

    Article  CAS  PubMed  Google Scholar 

  22. Mo J, Tymiak AA, Chen G (2013) Characterization of disulfide linkages in recombinant human granulocyte-colony stimulating factor. Rapid Commun Mass Spectrom 27(9):940–946. doi:10.1002/rcm.6530

    Article  CAS  PubMed  Google Scholar 

  23. Wu SL, Jiang H, Lu Q, Dai S, Hancock WS, Karger BL (2009) Mass spectrometric determination of disulfide linkages in recombinant therapeutic proteins using online LC-MS with electron-transfer dissociation. Anal Chem 81(1):112–122. doi:10.1021/ac801560k

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, 312 College Dr, Cincinnati, OH, 45221, USA

    Balasubrahmanyam Addepalli

Authors
  1. Balasubrahmanyam Addepalli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Balasubrahmanyam Addepalli .

Editor information

Editors and Affiliations

  1. Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky, USA

    Arthur G. Hunt

  2. Department of Biology, Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, College of the Environment and Ecology, Miami University, Oxford, OH, USA and Xiamen University, Xiamen, Fujian, Fujian, China

    Qingshun Quinn Li

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Addepalli, B. (2015). Detection of Disulfide Linkage by Chemical Derivatization and Mass Spectrometry. In: Hunt, A., Li, Q. (eds) Polyadenylation in Plants. Methods in Molecular Biology, vol 1255. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2175-1_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2175-1_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2174-4

  • Online ISBN: 978-1-4939-2175-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation