An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides

  • Sergey S. Zhokhov
  • Sergey V. Kovalyov
  • Tatiana Yu. Samgina
  • Albert T. Lebedev
Research Article


An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination.

Graphical Abstract


Leucine/isoleucine differentiation Orbitrap EThcD Tryptic peptides Peptide sequencing 



The authors are thankful to Thermo Fisher Scientific Inc., Textronica AG group (Moscow, Russia), and personally to Professor Alexander Makarov for providing Orbitrap Elite mass spectrometer for this work. They also express their gratitude to Professor Valery Shevchenko (Cancer Research Center, Moscow), Dr. Irina Tarasova (Insitute for Energy Problems of Chemical Physics, Russian Academy of Sciences), Dr. Ludmila Alekseeva and Dr. Lidia Kurochkina (Institute of Bioorganic Chemistry, Russ. Acad. Sci.) for proteins and synthetic peptides used in this studies.

Supplementary material

13361_2017_1674_MOESM1_ESM.doc (1.7 mb)
ESM 1 (DOC 1.67 MB)


  1. 1.
    Edman, P.: A method for the determination of the amino acid sequence in peptides. Arch. Biochem. 22, 475–476 (1949)Google Scholar
  2. 2.
    Edman, P., Begg, G.: A protein sequenator. Eur. J. Biochem. 1, 80–91 (1967)CrossRefGoogle Scholar
  3. 3.
    Artemenko, K.A., Zubarev, A.R., Samgina, T.Y., Lebedev, A.T., Savitski, M.M., Zubarev, R.A.: Two-dimensional mass mapping as a general method of data representation in comprehensive analysis of complex molecular mixtures. Anal. Chem. 81, 3738–3745 (2009)CrossRefGoogle Scholar
  4. 4.
    McLafferty, F.W., Turecek, F.: Interpretation of mass spectra, 4th edn. University Science Books, Mill Valley, CA (1993)Google Scholar
  5. 5.
    Johnson, R.S., Martin, S.A., Biemann, K., Stults, J.T., Watson, J.T.: Novel fragmentation process of peptides by collision-induced decomposition in a tandem mass spectrometer: differentiation of leucine and isoleucine. Anal. Chem. 59, 2621–2625 (1987)CrossRefGoogle Scholar
  6. 6.
    Martin, S.A., Biemann, K.: Mass spectrometric determination of the amino acid sequence of peptides and proteins. Mass Spectrom. Rev. 6, 1–78 (1987)CrossRefGoogle Scholar
  7. 7.
    Ramsey, S.L., Steinbornen, S.T., Waugh, R.J., Dua, S., Bowie, J.H.: A simple method for differentiating Leu and Ile in peptides. The negative‐ion mass spectra of M-h.− ions of phenylthiohydantoin Leu and Ile. Rapid Commun. Mass Spectrom. 9, 1241–1243 (1995)CrossRefGoogle Scholar
  8. 8.
    Tao, W.A., Wu, L., Cooks, R.G.: Differentiation and quantitation of isomeric dipeptides by low-energy dissociation of copper(II)-bound complexes. J. Am. Soc. Mass Spectrom. 12, 490–496 (2001)Google Scholar
  9. 9.
    Nakamura, T., Nagaki, H., Ohki, Y., Kinoshita, T.: Differentiation of leucine and isoleucine residues in peptides by consecutive reaction mass spectrometry. Anal. Chem. 62, 311–313 (1990)CrossRefGoogle Scholar
  10. 10.
    Armirotti, A., Millo, E., Damonte, G.: How to discriminate between leucine and isoleucine by low energy ESI-TRAP MSn. J. Am. Soc. Mass Spectrom. 18, 57–63 (2007)CrossRefGoogle Scholar
  11. 11.
    Xiao, Y., Vecchi, M.M., Wen, D.: Distinguishing between leucine and isoleucine by integrated LC-MS analysis using an Orbitrap Fusion mass spectrometer. Anal. Chem. 88, 10757–10766 (2016)CrossRefGoogle Scholar
  12. 12.
    Seymour, J.L., Turecek, F.: Distinction and quantitation of leucine-isoleucine isomers and lysine-glutamine isobars by electrospray ionization tandem mass spectrometry (MS(n), n = 2, 3) of copper(II)-diimine complexes. J. Mass Spectrom. 35, 566–571 (2000)CrossRefGoogle Scholar
  13. 13.
    Kjeldsen, F., Haselmann, K.F., Sørensen, E.S., Zubarev, R.A.: Distinguishing of Ile/Leu amino acid residues in the PP3 protein by (hot) electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 75, 1267–1274 (2003)CrossRefGoogle Scholar
  14. 14.
    Fung, Y.M., Chan, T.W.: Experimental and theoretical investigations of the loss of amino acid side chains in electron capture dissociation of model peptides. J. Am. Soc. Mass Spectrom. 16, 1523–1535 (2005)CrossRefGoogle Scholar
  15. 15.
    Han, H., Xia, Y., McLuckey, S.A.: Ion trap collisional activation of c- and z-ions formed via gas-phase ion/ion electron-transfer dissociation. J. Proteome Res. 6, 3062–3069 (2007)CrossRefGoogle Scholar
  16. 16.
    Gupta, K., Kumar, M., Chandrashekara, K., Krishnan, K.S., Balaram, P.: Combined electron transfer dissociation-collision-induced dissociation fragmentation in the mass spectrometric distinction of leucine, isoleucine, and hydroxyproline residues in peptide natural products. J. Proteome Res. 11, 515–522 (2012)CrossRefGoogle Scholar
  17. 17.
    Cook, S.L., Collin, O.L., Jackson, G.P.: Metastable atom‐activated dissociation mass spectrometry: leucine/isoleucine differentiation and ring cleavage of proline residues. J. Mass Spectrom. 44, 1211–1223 (2009)CrossRefGoogle Scholar
  18. 18.
    Lebedev, A.T., Damoc, E., Makarov, A.A., Samgina, T.Y.: Discrimination of leucine and isoleucine in peptides sequencing with Orbitrap Fusion mass spectrometer. Anal. Chem. 86, 7017–7022 (2014)CrossRefGoogle Scholar
  19. 19.
    Leymarie, N., Costello, C.E., O'Connor, P.B.: Electron capture dissociation initiates a free radical reaction cascade. J. Am. Chem. Soc. 125, 8949–8958 (2003)CrossRefGoogle Scholar
  20. 20.
    O’Connor, P.B., Lin, C., Cournoyer, J.J., Pittman, J.L., Belyayev, M., Budnik, B.A.: Long-lived electron capture dissociation product ions experience radical migration via hydrogen abstraction. J. Am. Soc. Mass Spectrom. 17, 576–585 (2006)CrossRefGoogle Scholar
  21. 21.
    Li, X., Lin, C., Han, L., Costello, C.E., O'Connor, P.B.: Charge remote fragmentation in electron capture and electron transfer dissociation. J. Am. Soc. Mass Spectrom. 21, 646–656 (2010)CrossRefGoogle Scholar
  22. 22.
    Bagal, D., Kast, E., Cao, P.: Rapid distinction of leucine and isoleucine in monoclonal antibodies using nanoflow LCMSn. Anal. Chem. (2016). doi: 10.1021/acs.analchem.6b03261 Google Scholar
  23. 23.
    Frese, C.K., Altelaar, A.F., van den Toorn, H., Nolting, D., Griep-Raming, J., Heck, A.J., Mohammed, S.: Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and higher-energy collision dissociation tandem mass spectrometry. Anal. Chem. 84, 9668–9673 (2012)CrossRefGoogle Scholar
  24. 24.
    Samgina, T.Y., Artemenko, K.A., Gorshkov, V.A., Nielsen, M.L., Savitski, M.M., Zubarev, R.A., Lebedev, A.T.: ESI MS/MS sequencing of novel skin peptides from Ranid frogs containing disulfide bridges. Eur. J. Mass Spectrom. 13, 155–163 (2007)CrossRefGoogle Scholar
  25. 25.
    Kurochkina, L.P., Semenyuk, P.I., Orlov, V.N., Robben, J., Sykilinda, N.N., Mesyanzhinov, V.V.: Expression and functional characterization of the first bacteriophage-encoded chaperonin. J. Virol. 86, 10103–10111 (2012)CrossRefGoogle Scholar
  26. 26.
    Swaney, D.L., McAlister, G.C., Wirtala, M., Schwartz, J.C., Syka, J.E., Coon, J.J.: Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 79, 477–485 (2007)CrossRefGoogle Scholar
  27. 27.
    Wysocki, V.H., Tsaprailis, G., Smith, L.L., Breci, L.A.: Mobile and localized protons: a framework for understanding peptide dissociation. J. Mass Spectrom. 35, 1399–1406 (2000)CrossRefGoogle Scholar
  28. 28.
    Samgina, T.Y., Artemenko, K.A., Gorshkov, V.A., Ogourtsov, S.V., Zubarev, R.A., Lebedev, A.T.: Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region. Rapid Commun. Mass Spectrom 23, 1241–1248 (2009)CrossRefGoogle Scholar
  29. 29.
    Samgina, T.Y., Kovalev, S.V., Gorshkov, V.A., Artemenko, K.A., Poljakov, N.B., Lebedev, A.T.: N-terminal tagging strategy for de novo sequencing of short peptides by ESI-MS/MS and MALDI-MS/MS. J. Am. Soc. Mass Spectrom. 21, 104–111 (2010)CrossRefGoogle Scholar
  30. 30.
    Price, W.D., Schnier, P.D., Jockusch, R.A., Strittmatter, E.F., Williams, E.R.: Unimolecular reaction kinetics in the high-pressure limit without collisions. J. Am. Chem. Soc. 118, 10640–10644 (1996)CrossRefGoogle Scholar
  31. 31.
    Laskin, J., Futrell, J.H.: Activation of large ions in FT-ICR mass spectrometry. Mass Spectrom. Rev. 24, 135–167 (2005)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2017

Authors and Affiliations

  • Sergey S. Zhokhov
    • 1
  • Sergey V. Kovalyov
    • 1
  • Tatiana Yu. Samgina
    • 1
  • Albert T. Lebedev
    • 1
  1. 1.Department of ChemistryM.V. Lomonosov Moscow State UniversityMoscowRussia

Personalised recommendations