Skip to main content

Identification of Arginylated Proteins by Mass Spectrometry

  • Protocol
Protein Arginylation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1337))

Abstract

Here we describe the method for identification of arginylated proteins by mass spectrometry. This method has been originally applied to the identification of N-terminally added Arg on proteins and peptides, and then expanded to identification of side chain arginylation which has been recently described by our groups. The key steps in this method include the use of the mass spectrometry instruments that can identify peptides with very high pass accuracy (Orbitrap) and apply stringent mass cutoffs during automated data analysis, followed by manual validation of the identified spectra. These methods can be used with both complex and purified protein samples and, to date, constitute the only reliable way to confirm arginylation at a particular site on a protein or peptide.

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

Access this chapter

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. Kaji H, Novelli GD, Kaji A (1963) A soluble amino acid-incorporating system from rat liver. Biochim Biophys Acta 76:474–477

    Article  CAS  PubMed  Google Scholar 

  2. Kaji A, Kaji H, Novelli GD (1963) A soluble amino acid incorporating system. Biochem Biophys Res Commun 10:406–409

    Article  CAS  PubMed  Google Scholar 

  3. Kopitz J, Rist B, Bohley P (1990) Post-translational arginylation of ornithine decarboxylase from rat hepatocytes. Biochem J 267(2):343–348

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Bohley P, Kopitz J, Adam G (1988) Surface hydrophobicity, arginylation and degradation of cytosol proteins from rat hepatocytes. Biol Chem Hoppe Seyler 369(Suppl):307–310

    CAS  PubMed  Google Scholar 

  5. Bohley P, Kopitz J, Adam G (1988) Arginylation, surface hydrophobicity and degradation of cytosol proteins from rat hepatocytes. Adv Exp Med Biol 240:159–169

    Article  CAS  PubMed  Google Scholar 

  6. Ciechanover A, Ferber S, Ganoth D, Elias S, Hershko A, Arfin S (1988) Purification and characterization of arginyl-tRNA-protein transferase from rabbit reticulocytes. Its involvement in post-translational modification and degradation of acidic NH2 termini substrates of the ubiquitin pathway. J Biol Chem 263(23):11155–11167

    CAS  PubMed  Google Scholar 

  7. Soffer RL (1971) Enzymatic modification of proteins. 4. Arginylation of bovine thyroglobulin. J Biol Chem 246(5):1481–1484

    CAS  PubMed  Google Scholar 

  8. Soffer RL (1975) Enzymatic arginylation of beta-melanocyte-stimulating hormone and of angiotensin II. J Biol Chem 250(7):2626–2629

    CAS  PubMed  Google Scholar 

  9. Zhang N, Donnelly R, Ingoglia NA (1998) Evidence that oxidized proteins are substrates for N-terminal arginylation. Neurochem Res 23(11):1411–1420

    Article  CAS  PubMed  Google Scholar 

  10. Wong CC, Xu T, Rai R, Bailey AO, Yates JR III, Wolf YI, Zebroski H, Kashina A (2007) Global analysis of posttranslational protein arginylation. PLoS Biol 5(10):e258

    Article  PubMed Central  PubMed  Google Scholar 

  11. Rai R, Wong CC, Xu T, Leu NA, Dong DW, Guo C, McLaughlin KJ, Yates JR III, Kashina A (2008) Arginyltransferase regulates alpha cardiac actin function, myofibril formation and contractility during heart development. Development 135(23):3881–3889

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Cornachione AS, Leite FS, Wang J, Leu NA, Kalganov A, Volgin D, Han X, Xu T, Cheng YS, Yates JR III, Rassier DE, Kashina A (2014) Arginylation of myosin heavy chain regulates skeletal muscle strength. Cell Rep 8(2):470–476

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Lian L, Suzuki A, Hayes V, Saha S, Han X, Xu T, Yates JR III, Poncz M, Kashina A, Abrams CS (2014) Loss of ATE1-mediated arginylation leads to impaired platelet myosin phosphorylation, clot retraction, and in vivo thrombosis formation. Haematologica 99(3):554–560

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Saha S, Wong CC, Xu T, Namgoong S, Zebroski H, Yates JR III, Kashina A (2011) Arginylation and methylation double up to regulate nuclear proteins and nuclear architecture in vivo. Chem Biol 18(11):1369–1378

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Xu T, Wong CC, Kashina A, Yates JR III (2009) Identification of N-terminally arginylated proteins and peptides by mass spectrometry. Nat Protoc 4(3):325–332

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Xu T, Venable JD, Park SK, Cociorva D, Lu B, Liao L, Wohlschlegel J, Hewel J, Yates JR III (2006) ProLuCID, a fast and sensitive tandem mass spectra-based protein identification program. Mol Cell Proteomics 5(10):S174

    Google Scholar 

  17. Shen Y, Tolic N, Hixson KK, Purvine SO, Pasa-Tolic L, Qian WJ, Adkins JN, Moore RJ, Smith RD (2008) Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags. Anal Chem 80(6):1871–1882

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Tabb DL, McDonald WH, Yates JR III (2002) DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J Proteome Res 1(1):21–26

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Cociorva D, L Tabb D, Yates JR (2007) Validation of tandem mass spectrometry database search results using DTASelect. Curr Protoc Bioinformatics Chapter 13:Unit 13.4

    Google Scholar 

  20. Karakozova M, Kozak M, Wong CC, Bailey AO, Yates JR III, Mogilner A, Zebroski H, Kashina A (2006) Arginylation of beta-actin regulates actin cytoskeleton and cell motility. Science 313(5784):192–196

    Article  CAS  PubMed  Google Scholar 

  21. Tsaprailis G, Nair H, Somogyi A, Wysocki V, Zhong W, Futrell J, Summerfield S, Gaskell SJ (1999) Influence of secondary structure on the fragmentation of protonated peptides. J Am Chem Soc 121(22):5142–5154

    Article  CAS  Google Scholar 

  22. Dongre A, Jones J, Somogyi A, Wysocki V (1996) Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency: evidence for the mobile proton model. J Am Chem soc 118(35):8365–8374

    Article  CAS  Google Scholar 

  23. Washburn MP, Wolters D, Yates JR (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19(3):242–247

    Article  CAS  PubMed  Google Scholar 

  24. Eng JK, McCormack AL, Yates JR III (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989

    Article  CAS  PubMed  Google Scholar 

  25. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18):3551–3567

    Article  CAS  PubMed  Google Scholar 

  26. Craig R, Beavis RC (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20(9):1466–1467

    Article  CAS  PubMed  Google Scholar 

  27. Geer LY, Markey SP, Kowalak JA, Wagner L, Xu M, Maynard DM, Yang X, Shi W, Bryant SH (2004) Open mass spectrometry search algorithm. J Proteome Res 3(5):958–964

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to John R. Yates III .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kashina, A.S., Yates, J.R. (2015). Identification of Arginylated Proteins by Mass Spectrometry. In: Kashina, A. (eds) Protein Arginylation. Methods in Molecular Biology, vol 1337. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2935-1_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2935-1_13

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2934-4

  • Online ISBN: 978-1-4939-2935-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics