Discrimination of Isobaric Leu/Ile Residues by MALDI In-Source Decay Mass Spectrometry

  • Daiki AsakawaEmail author
  • Nicolas Smargiasso
  • Edwin De Pauw
Application Note


MALDI in-source decay (ISD) has been used for top-down sequencing of proteins. The use of the matrix 1,5-diaminonapthalene (1,5-DAN) gave abundant w ions, which are formed from the unimolecular dissociation of z• radical fragments via α cleavage reaction and thus help identify which of the isobaric amino acids, Leu or Ile, is present. The high abundance of w ions in MALDI-ISD with 1,5-DAN results from the low collision rate in the MALDI plume. MALDI-ISD with 1,5-DAN appears to be an useful method for the top-down sequencing of proteins, including discrimination of Leu and Ile near the C-terminal end.

Key words

Initial velocity Side-chain loss 1,5-Diaminonapthalene Negative-ion mode Top-down sequencing 



D.A. acknowledges a research fellowship from the Japan Society for the Promotion of Science for Young Scientists (grant 23-10272). N.S. is a FRS-FNRS logistics collaborator. Walloon Region and FNRS contributed to Mass Spectrometry platform funding.

Supplementary material

13361_2012_523_MOESM1_ESM.doc (944 kb)
ESM 1 (DOC 944 kb)


  1. 1.
    Paizs, B., Suhai, S.: Fragmentation pathways of protonated peptides. Mass Spectrom. Rev. 24, 508–548 (2005)CrossRefGoogle Scholar
  2. 2.
    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
  3. 3.
    Zubarev, R.A.: Reactions of polypeptide ions with electrons in the gas phase. Mass Spectrom. Rev. 22, 57–77 (2003)CrossRefGoogle Scholar
  4. 4.
    Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanovitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 101, 9528–9533 (2004)CrossRefGoogle Scholar
  5. 5.
    Kjeldsen, F., Haselmann, K.F., Sørensen, E.S., Zubarev, R.A.: Distinguishing of Ile/Leu amino acid residues in the pp 3 protein by (hot) electron capture dissociation in fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 75, 1267–1274 (2003)CrossRefGoogle Scholar
  6. 6.
    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)Google Scholar
  7. 7.
    Chung, T.W., Tureček, F.: Backbone and side-chain specific dissociations of z ions from non-tryptic peptides. J. Am. Soc. Mass Spectrom. 21, 1279–1295 (2010)CrossRefGoogle Scholar
  8. 8.
    Takayama, M.: N–Cα bond cleavage of the peptide backbone via hydrogen abstraction. J. Am. Soc. Mass Spectrom. 12, 1044–1049 (2001)CrossRefGoogle Scholar
  9. 9.
    Köcher, T., Engström, Å., Zubarev, R.A.: Fragmentation of peptides in MALDI in-source decay mediated by hydrogen radicals. Anal. Chem. 77, 172–177 (2005)CrossRefGoogle Scholar
  10. 10.
    Hardouin, J.: Protein sequence information by matrix-assisted laser desorption/ionization in-source decay mass spectrometry. Mass Spectrom. Rev. 26, 672–682 (2007)CrossRefGoogle Scholar
  11. 11.
    Sellami, L., Belgacem, O., Villard, C., Openshaw, M.E., Barbier, P., Lafitte, D.: In-source decay and pseudo tandem mass spectrometry fragmentation processes of entire high mass proteins on a hybrid vacuum matrix-assisted laser desorption ionization-quadrupole ion-trap time-of-flight mass spectrometer. Anal. Chem. 84, 5180–5185 (2012)CrossRefGoogle Scholar
  12. 12.
    Soltwisch, J., Dreisewerd, K.: Discrimination of isobaric leucine and isoleucine residues and analysis of post-translational modifications in peptides by MALDI in-source decay mass spectrometry combined with collisional cooling. Anal. Chem. 82, 5628–5635 (2010)CrossRefGoogle Scholar
  13. 13.
    Demeure, K., Quinton, L., Gabelica, V., De Pauw, E.: Rational selection of the optimum MALDI matrix for top-down proteomics by in-source decay. Anal. Chem. 79, 8678–8685 (2007)CrossRefGoogle Scholar
  14. 14.
    Smargiasso, N., Quinton, L., De Pauw, E.: 2-Aminobenzamide and 2-aminobenzoic acid as new MALDI matrices inducing radical mediated in-source decay of peptides and proteins. J. Am. Soc. Mass Spectrom. 23, 469–474 (2012)CrossRefGoogle Scholar
  15. 15.
    Demeure, K., Gabelica, V., De Pauw, E.: New advances in the understanding of the in-source decay fragmentation of peptides in MALDI-TOF-MS. J. Am. Soc. Mass Spectrom. 21, 1906–1917 (2010)Google Scholar
  16. 16.
    Spengler, B., Kirsch, D.: On the formation of initial ion velocities in matrix-assisted laser desorption ionization: Virtual desorption time as an additional parameter describing ion ejection dynamics. Int. J. Mass Spectrom. 226, 71–83 (2003)CrossRefGoogle Scholar
  17. 17.
    Juhasz, P., Vestal, M.L., Martin, S.A.: On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of delayed extraction time-of-flight mass spectra. J. Am. Soc. Mass Spectrom. 8, 209–217 (1997)CrossRefGoogle Scholar
  18. 18.
    Asakawa, D., Takayama, M.: Cα–C bond cleavage of the peptide backbone in MALDI in-source decay using salicylic acid derivative matrices. J. Am. Soc. Mass Spectrom. 22, 1224–1233 (2011)CrossRefGoogle Scholar
  19. 19.
    Asakawa, D., Takayama, M.: Fragmentation processes of hydrogen-deficient peptide radicals in matrix-assisted laser desorption/ionization in-source decay mass spectrometry. J. Phys. Chem. B 116, 4016–4023 (2012)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2013

Authors and Affiliations

  • Daiki Asakawa
    • 1
    • 2
    Email author
  • Nicolas Smargiasso
    • 1
  • Edwin De Pauw
    • 1
  1. 1.Chemistry Department and GIGA-R, Mass Spectrometry LaboratoryUniversity of LiegeLiegeBelgium
  2. 2.Graduate School of Nano Bioscience, Mass Spectrometry LaboratoryYokohama City UniversityYokohamaJapan

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