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Identification of Arginylated Proteins by Mass Spectrometry

  • Anna S. Kashina
  • John R. YatesIIIEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Posttranslational modifications Protein arginylation Mass spectrometry 

References

  1. 1.
    Kaji H, Novelli GD, Kaji A (1963) A soluble amino acid-incorporating system from rat liver. Biochim Biophys Acta 76:474–477CrossRefPubMedGoogle Scholar
  2. 2.
    Kaji A, Kaji H, Novelli GD (1963) A soluble amino acid incorporating system. Biochem Biophys Res Commun 10:406–409CrossRefPubMedGoogle Scholar
  3. 3.
    Kopitz J, Rist B, Bohley P (1990) Post-translational arginylation of ornithine decarboxylase from rat hepatocytes. Biochem J 267(2):343–348PubMedCentralCrossRefPubMedGoogle Scholar
  4. 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):307–310PubMedGoogle Scholar
  5. 5.
    Bohley P, Kopitz J, Adam G (1988) Arginylation, surface hydrophobicity and degradation of cytosol proteins from rat hepatocytes. Adv Exp Med Biol 240:159–169CrossRefPubMedGoogle Scholar
  6. 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):11155–11167PubMedGoogle Scholar
  7. 7.
    Soffer RL (1971) Enzymatic modification of proteins. 4. Arginylation of bovine thyroglobulin. J Biol Chem 246(5):1481–1484PubMedGoogle Scholar
  8. 8.
    Soffer RL (1975) Enzymatic arginylation of beta-melanocyte-stimulating hormone and of angiotensin II. J Biol Chem 250(7):2626–2629PubMedGoogle Scholar
  9. 9.
    Zhang N, Donnelly R, Ingoglia NA (1998) Evidence that oxidized proteins are substrates for N-terminal arginylation. Neurochem Res 23(11):1411–1420CrossRefPubMedGoogle Scholar
  10. 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):e258PubMedCentralCrossRefPubMedGoogle Scholar
  11. 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):3881–3889PubMedCentralCrossRefPubMedGoogle Scholar
  12. 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):470–476PubMedCentralCrossRefPubMedGoogle Scholar
  13. 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):554–560PubMedCentralCrossRefPubMedGoogle Scholar
  14. 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):1369–1378PubMedCentralCrossRefPubMedGoogle Scholar
  15. 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):325–332PubMedCentralCrossRefPubMedGoogle Scholar
  16. 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):S174Google Scholar
  17. 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):1871–1882PubMedCentralCrossRefPubMedGoogle Scholar
  18. 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):21–26PubMedCentralCrossRefPubMedGoogle Scholar
  19. 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 13.4Google Scholar
  20. 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):192–196CrossRefPubMedGoogle Scholar
  21. 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):5142–5154CrossRefGoogle Scholar
  22. 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):8365–8374CrossRefGoogle Scholar
  23. 23.
    Washburn MP, Wolters D, Yates JR (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19(3):242–247CrossRefPubMedGoogle Scholar
  24. 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 5:976–989CrossRefPubMedGoogle Scholar
  25. 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):3551–3567CrossRefPubMedGoogle Scholar
  26. 26.
    Craig R, Beavis RC (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20(9):1466–1467CrossRefPubMedGoogle Scholar
  27. 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):958–964CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Animal Biology, School of Veterinary MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Department of Chemical PhysiologyThe Scripps Research InstituteLa JollaUSA

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