Fragmentation of Multi-charged Derivatized Lysine Using Nanospray CID Tandem Mass Spectrometry

  • Tianjiao Huang
  • Jordan M. Rabus
  • Benjamin J. Bythell
  • James L. EdwardsEmail author
Short Communication


We demonstrate increasing the charge state of small molecules using derivatized lysine as our model system. Lysine is chemically tagged with three tertiary amines which enables efficient production of highly charged analytes. A +3 charge state is obtained from direct infusion nanoelectrospray conditions. Collisional activation of the +3 derivatized lysine yielded structurally informative product ions corresponding to cleavages across the analyte backbone and within the proton affinity tags. This suggests a role for multi-charging of metabolites in both targeted MRM analyses and untargeted analyses to help identify novel metabolites. Density functional calculations aid peak assignment and rationalization of structure-property relationships.

Graphical Abstract


Supercharging Nanospray Metabolite ETC CID 



This material is based upon work supported by the National Science Foundation under grant no. DBI-0922879 for acquisition of the LTQ-Velos Pro Orbitrap LC-MS/MS. Brad Evans is thanked for his assistance with the ETD experiments.

Supplementary material

13361_2019_2154_MOESM1_ESM.pdf (1.3 mb)
ESM 1 (PDF 1.30 mb)


  1. 1.
    Frey, B.L., Ladror, D.T., Sondalle, S.B., Krusemark, C.J., Jue, A.L., Coon, J.J., Smith, L.M.: Chemical derivatization of peptide carboxyl groups for highly efficient electron transfer dissociation. J. Am. Soc. Mass Spectrom. 24, 1710–1721 (2013)CrossRefGoogle Scholar
  2. 2.
    Iavarone, A.T., Williams, E.R.: Mechanism of charging and supercharging molecules in electrospray ionization. J. Am. Chem. Soc. 125, 2319–2327 (2003)CrossRefGoogle Scholar
  3. 3.
    Iavarone, A.T., Jurchen, J.C., Williams, E.R.: Supercharged protein and peptide ions formed by electrospray ionization. Anal. Chem. 73, 1455–1460 (2001)CrossRefGoogle Scholar
  4. 4.
    Riley, N.M., Coon, J.J.: The role of electron transfer dissociation in modern proteomics. Anal. Chem. 90, 40–64 (2018)CrossRefGoogle Scholar
  5. 5.
    Li, Y., Cole, R.B.: Shifts in peptide and protein charge state distributions with varying spray tip orifice diameter in nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 75, 5739–5746 (2003)CrossRefGoogle Scholar
  6. 6.
    Ogorzalek Loo, R.R., Lakshmanan, R., Loo, J.A.: What protein charging (and supercharging) reveal about the mechanism of electrospray ionization. J. Am. Soc. Mass Spectrom. 25, 1675–1693 (2014)CrossRefGoogle Scholar
  7. 7.
    Wilm, M., Mann, M.: Analytical properties of the nanoelectrospray ion source. Anal. Chem. 68, 1–8 (1996)CrossRefGoogle Scholar
  8. 8.
    Ahadi, E., Konermann, L.: Surface charge of electrosprayed water nanodroplets: a molecular dynamics study. J. Am. Chem. Soc. 132, 11270–11277 (2010)CrossRefGoogle Scholar
  9. 9.
    Cassou, C.A., Sterling, H.J., Susa, A.C., Williams, E.R.: Electrothermal supercharging in mass spectrometry and tandem mass spectrometry of native proteins. Anal. Chem. 85, 138–146 (2013)CrossRefGoogle Scholar
  10. 10.
    Geromanos, S., Freckleton, G., Tempst, P.: Tuning of an electrospray ionization source for maximum peptide-ion transmission into a mass spectrometer. Anal. Chem. 72, 777–790 (2000)CrossRefGoogle Scholar
  11. 11.
    Heemskerk, A.A., Busnel, J.M., Schoenmaker, B., Derks, R.J., Klychnikov, O., Hensbergen, P.J., Deelder, A.M., Mayboroda, O.A.: Ultra-low flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics. Anal. Chem. 84, 4552–4559 (2012)CrossRefGoogle Scholar
  12. 12.
    Marginean, I., Kelly, R.T., Prior, D.C., LaMarche, B.L., Tang, K., Smith, R.D.: Analytical characterization of the electrospray ion source in the nanoflow regime. Anal. Chem. 80, 6573–6579 (2008)CrossRefGoogle Scholar
  13. 13.
    Yuill, E.M., Sa, N., Ray, S.J., Hieftje, G.M., Baker, L.A.: Electrospray ionization from nanopipette emitters with tip diameters of less than 100 nm. Anal. Chem. 85, 8498–8502 (2013)CrossRefGoogle Scholar
  14. 14.
    Huang, T., Armbruster, M., Lee, R., Hui, D.S., Edwards, J.L.: Metabolomic analysis of mammalian cells and human tissue through one-pot two stage derivatizations using sheathless capillary electrophoresis-electrospray ionization-mass spectrometry. J. Chromatogr. A. 1567, 219–225 (2018)CrossRefGoogle Scholar
  15. 15.
    Huang, T., Armbruster, M.R., Coulton, J.B., Edwards, J.L.: Chemical tagging in mass spectrometry for systems biology. Anal. Chem. (2018)Google Scholar
  16. 16.
    Huang, T., Toro, M., Lee, R., Hui, D.S., Edwards, J.L.: Multi-functional derivatization of amine, hydroxyl, and carboxylate groups for metabolomic investigations of human tissue by electrospray ionization mass spectrometry. Analyst. 143, 3408–3414 (2018)CrossRefGoogle Scholar
  17. 17.
    Moss, C.L., Liang, W., Li, X., Turecek, F.: The early life of a peptide cation-radical. Ground and excited-state trajectories of electron-based peptide dissociations during the first 330 femtoseconds. J. Am. Soc. Mass Spectrom. 23, 446–459 (2012)CrossRefGoogle Scholar
  18. 18.
    Wu, S.L., Huhmer, A.F., Hao, Z., Karger, B.L.: On-line LC-MS approach combining collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced species for the trace-level characterization of proteins with post-translational modifications. J. Proteome Res. 6, 4230–4244 (2007)CrossRefGoogle Scholar
  19. 19.
    Yin, S., Loo, J.A.: Top-down mass spectrometry of supercharged native protein-ligand complexes. Int. J. Mass Spectrom. 300, 118–122 (2011)CrossRefGoogle Scholar
  20. 20.
    Bythell, B.J., Somogyi, A., Paizs, B.: What is the structure of b(2) ions generated from doubly protonated tryptic peptides? J. Am. Soc. Mass Spectrom. 20, 618–624 (2009)CrossRefGoogle Scholar
  21. 21.
    Bythell, B.J., Suhai, S., Somogyi, A., Paizs, B.: Proton-driven amide bond-cleavage pathways of gas-phase peptide ions lacking mobile protons. J. Am. Chem. Soc. 131, 14057–14065 (2009)CrossRefGoogle Scholar
  22. 22.
    Wysocki, V.H., Tsaprailis, G., Smith, L.L., Breci, L.A.: Special feature: commentary - mobile and localized protons: a framework for understanding peptide dissociation. J. Mass Spectrom. 35, 1399–1406 (2000)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistrySaint Louis UniversitySt LouisUSA
  2. 2.Department of Chemistry and BiochemistryUniversity of Missouri St LouisSt LouisUSA

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