Analytical and Bioanalytical Chemistry

, Volume 405, Issue 29, pp 9321–9331 | Cite as

Specific biotinylation and sensitive enrichment of citrullinated peptides

  • Astrid E. V. TutturenEmail author
  • Anders Holm
  • Burkhard Fleckenstein
Research Paper


Protein citrullination is a posttranslational modification where peptidylarginine is enzymatically deiminated to form peptidylcitrulline. Although the role of protein citrullination in both health and disease is being increasingly recognised, techniques available to identify citrullinated proteins and to map their citrullination site(s) are rare and often show poor sensitivity. Here, we present a sensitive technique for specific modification and selective enrichment of citrullinated peptides from complex biological samples. The technique is based on highly specific in-solution biotinylation of citrulline residues followed by selective enrichment of modified peptides using streptavidin beads. We demonstrate that a synthetic citrulline-containing peptide can be selectively enriched when less than 0.5 pmol is spiked into a highly heterogeneous peptide mixture. After enrichment, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of an aliquot of the streptavidin eluate corresponding to theoretically 50 fmol of the spiked-in peptide showed a prominent signal. We further demonstrate the sensitivity of our technique by enrichment of citrullinated peptides from enzymatically deiminated myelin basic protein (MBP), when 10 pmol was spiked into a heterogeneous biological digest. In MALDI-TOF MS analysis, six MBP-derived citrullinated peptides were observed, showing the efficiency of this enrichment strategy. The high sensitivity combined with the remarkable specificity of the described technique makes it a valuable tool for elucidating citrullination in various biological processes.


Schematic view of the established technique for modification and enrichment of citrullinated peptides (top). Enrichment of the synthetic peptide RPSQ-Cit-HGSK (0.5 pmol) from a complex sample (8.2 nmol) (bottom). After enrichment an amount corresponding to 50 fmol of the spiked-in peptide was analysed and is observed as a prominent signal (m/z 1569.85)


Protein citrullination Chemical modification Biotinylation Peptide enrichment Rheumatoid arthritis Mass spectrometry 



We thank Fridtjof Lund-Johansen for providing the A431 cell line and Maria Stensland for providing recombinant human PAD4.We further thank Dr. Gustavo de Souza for critical reading of the manuscript and discussions. Samples were analysed by A.E.V.T. at the Proteomics Core Facility, OUS-UiO, which is supported by an Infrastructure grant from the South-East Health Authority. This research was supported by grants from the MLSUiO-Molecular Life Science, University of Oslo to A.E.V.T. and from the Research Council of Norway to A.H. and B.F.


  1. 1.
    Vossenaar ER, Nijenhuis S, Helsen MM, van der Heijden A, Senshu T, van den Berg WB, van Venrooij WJ, Joosten LA (2003) Citrullination of synovial proteins in murine models of rheumatoid arthritis. Arthritis Rheum 48(9):2489–2500CrossRefGoogle Scholar
  2. 2.
    Tarcsa E, Marekov LN, Mei G, Melino G, Lee SC, Steinert PM (1996) Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin. J Biol Chem 271(48):30709–30716CrossRefGoogle Scholar
  3. 3.
    Gyorgy B, Toth E, Tarcsa E, Falus A, Buzas EI (2006) Citrullination: a posttranslational modification in health and disease. Int J Biochem Cell Biol 38(10):1662–1677CrossRefGoogle Scholar
  4. 4.
    Baka Z, Gyorgy B, Geher P, Buzas EI, Falus A, Nagy G (2012) Citrullination under physiological and pathological conditions. Joint Bone Spine 79(5):431–436CrossRefGoogle Scholar
  5. 5.
    Wegner N, Lundberg K, Kinloch A, Fisher B, Malmstrom V, Feldmann M, Venables PJ (2010) Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis. Immunol Rev 233(1):34–54CrossRefGoogle Scholar
  6. 6.
    Chapuy-Regaud S, Sebbag M, Baeten D, Clavel C, Foulquier C, De KF, Serre G (2005) Fibrin deimination in synovial tissue is not specific for rheumatoid arthritis but commonly occurs during synovitides. J Immunol 174(8):5057–5064Google Scholar
  7. 7.
    Moscarello MA, Wood DD, Ackerley C, Boulias C (1994) Myelin in multiple sclerosis is developmentally immature. J Clin Invest 94(1):146–154CrossRefGoogle Scholar
  8. 8.
    Ishigami A, Ohsawa T, Hiratsuka M, Taguchi H, Kobayashi S, Saito Y, Murayama S, Asaga H, Toda T, Kimura N, Maruyama N (2005) Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer’s disease. J Neurosci Res 80(1):120–128CrossRefGoogle Scholar
  9. 9.
    Chang X, Fang K (2010) PADI4 and tumourigenesis. Cancer Cell Int 10:7CrossRefGoogle Scholar
  10. 10.
    Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ (1998) Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest 101(1):273–281CrossRefGoogle Scholar
  11. 11.
    Schellekens GA, Visser H, de Jong BA, van den Hoogen FH, Hazes JM, Breedveld FC, van Venrooij WJ (2000) The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 43(1):155–163CrossRefGoogle Scholar
  12. 12.
    De Ceuleneer M, Van Steendam K, Dhaenens M, Deforce D (2012) In vivo relevance of citrullinated proteins and the challenges in their detection. Proteomics 12(6):752–760Google Scholar
  13. 13.
    Senshu T, Sato T, Inoue T, Akiyama K, Asaga H (1992) Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane. Anal Biochem 203(1):94–100CrossRefGoogle Scholar
  14. 14.
    Holm A, Rise F, Sessler N, Sollid LM, Undheim K, Fleckenstein B (2006) Specific modification of peptide-bound citrulline residues. Anal Biochem 352(1):68–76CrossRefGoogle Scholar
  15. 15.
    Makrygiannakis D, Hermansson M, Ulfgren AK, Nicholas AP, Zendman AJ, Eklund A, Grunewald J, Skold CM, Klareskog L, Catrina AI (2008) Smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in BAL cells. Ann Rheum Dis 67(10):1488–1492CrossRefGoogle Scholar
  16. 16.
    Matsuo K, Xiang Y, Nakamura H, Masuko K, Yudoh K, Noyori K, Nishioka K, Saito T, Kato T (2006) Identification of novel citrullinated autoantigens of synovium in rheumatoid arthritis using a proteomic approach. Arthritis Res Ther 8(6):R175CrossRefGoogle Scholar
  17. 17.
    Stensland M, Holm A, Kiehne A, Fleckenstein B (2009) Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry. Rapid Commun Mass Spectrom 23(17):2754–2762CrossRefGoogle Scholar
  18. 18.
    De Ceuleneer M, De Wit V, Van Steendam K, Van Nieuwerburgh F, Tilleman K, Deforce D (2011) Modification of citrulline residues with 2,3-butanedione facilitates their detection by liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 25(11):1536–1542CrossRefGoogle Scholar
  19. 19.
    Hao G, Wang D, Gu J, Shen Q, Gross SS, Wang Y (2008) Neutral loss of isocyanic acid in peptide CID spectra: a novel diagnostic marker for mass spectrometric identification of protein citrullination. J Am Soc Mass Spectrom 20(4):723–727CrossRefGoogle Scholar
  20. 20.
    Bicker KL, Subramanian V, Chumanevich AA, Hofseth LJ, Thompson PR (2012) Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination. J Am Chem Soc 134(41):17015–17018CrossRefGoogle Scholar
  21. 21.
    Choi M, Song JS, Kim HJ, Cha S, Lee EY (2013) Matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of peptide citrullination site using Br signature. Anal Biochem 437(1):62–67CrossRefGoogle Scholar
  22. 22.
    Tutturen AE, Holm A, Jorgensen M, Stadtmuller P, Rise F, Fleckenstein B (2010) A technique for the specific enrichment of citrulline-containing peptides. Anal Biochem 403(1–2):43–51CrossRefGoogle Scholar
  23. 23.
    Wagner R, Garrett RA (1978) A new RNA-RNA crosslinking reagent and its application to ribosomal 5S RNA. Nucleic Acids Res 5(11):4065–4075CrossRefGoogle Scholar
  24. 24.
    Cagney G, Amiri S, Premawaradena T, Lindo M, Emili A (2003) In silico proteome analysis to facilitate proteomics experiments using mass spectrometry. Proteome Sci 1(1):5–19CrossRefGoogle Scholar
  25. 25.
    Stensland ME, Pollmann S, Molberg O, Sollid LM, Fleckenstein B (2009) Primary sequence, together with other factors, influence peptide deimination by peptidylarginine deiminase-4. Biol Chem 390(2):99–107CrossRefGoogle Scholar
  26. 26.
    Gao Y, Wang Y (2006) Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal. Biochemistry 45(51):15654–15660CrossRefGoogle Scholar
  27. 27.
    Kinloch A, Tatzer V, Wait R, Peston D, Lundberg K, Donatien P, Moyes D, Taylor PC, Venables PJ (2005) Identification of citrullinated alpha-enolase as a candidate autoantigen in rheumatoid arthritis. Arthritis Res Ther 7(6):R1421–R1429CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Astrid E. V. Tutturen
    • 1
    Email author
  • Anders Holm
    • 2
  • Burkhard Fleckenstein
    • 1
  1. 1.Centre for Immune Regulation, Institute of ImmunologyUniversity of OsloOsloNorway
  2. 2.Centre for Immune Regulation, Institute of ImmunologyOslo University Hospital-RikshospitaletOsloNorway

Personalised recommendations