New reagents for increasing ESI multiple charging of proteins and protein complexes

Short Communication


The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am. Soc. Mass Spectrom.2009,20, 593–596) to enhance multiple charging of native proteins and noncovalent protein complexes in electrospray ionization (ESI) mass spectra. Additional new reagents have been found to “supercharge” proteins from nondenaturing solutions; several of these reagents are shown to be more effective than m-NBA for increasing positive charging. Using the myoglobin protein-protoporphyrin IX (heme) complex, the following reagents were shown to increase ESI charging: benzyl alcohol, m-nitroacetophenone, m-nitrobenzonitrile, o-NBA, m-NBA, p-NBA, m-nitrophenyl ethanol, sulfolane (tetramethylene sulfone), and m-(trifluoromethyl)-benzyl alcohol. Based on average charge state, sulfolane displayed a greater charge increase (61%) than m-NBA (21%) for myoglobin in aqueous solutions. The reagents that promote higher ESI charging appear to have low solution-phase basicities and relatively low gas-phase basicities, and are less volatile than water. Another feature of mass spectra from some of the active reagents is that adducts are present on higher charge states, suggesting that a mechanism by which proteins acquire additional charge involves direct interaction with the reagent, in addition to other factors such as surface tension and protein denaturation.

Supplementary material

13361_2011_210100127_MOESM1_ESM.pdf (196 kb)
Supplementary material, approximately 201 KB.


  1. 1.
    Mann, M.; Meng, C. K.; Fenn, J. B. Interpreting Mass Spectra of Multiply Charged Ions. Anal. Chem. 1989, 61, 1702–1708.CrossRefGoogle Scholar
  2. 2.
    Loo, J. A.; Edmonds, C. G.; Smith, R. D. Primary Sequence Information from Intact Proteins by Electrospray Ionization Tandem Mass Spectrometry. Science 1990, 248, 201–204.CrossRefGoogle Scholar
  3. 3.
    Loo, J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Collisional Activation and Dissociation of Large Multiply Charged Proteins Produced by Electrospray Ionization. Anal. Chim. Acta 1990, 241, 167–173.CrossRefGoogle Scholar
  4. 4.
    Loo, J. A.; Udseth, H. R.; Smith, R. D. Solvent Effects on the Charge Distribution Observed with Electrospray Ionization-Mass Spectrometry of Large Molecules. Biomed. Environ. Mass Spectrom. 1988, 17, 411–414.CrossRefGoogle Scholar
  5. 5.
    Iavarone, A. T.; Jurchen, J. C.; Williams, E. R. Supercharged Protein and Peptide Ions Formed by Electrospray Ionization. Anal. Chem. 2001, 73, 1455–1460.CrossRefGoogle Scholar
  6. 6.
    Iavarone, A. T.; Williams, E. R. Mechanism of Charging and SuperCharging Molecules in Electrospray Ionization. J. Am. Chem. Soc. 2003, 125, 2319–2327.CrossRefGoogle Scholar
  7. 7.
    Lomeli, S. H.; Yin, S.; Ogorzalek Loo, R. R.; Loo, J. A. Increasing Charge While Preserving Noncovalent Protein Complexes for ESI-MS. J. Am. Soc. Mass Spectrom. 2009, 20, 593–596.CrossRefGoogle Scholar
  8. 8.
    de la Mora, J. F. Electrospray Ionization of Large Multiply Charged Species Proceeds via Dole’s Charged Residue Mechanism. Anal. Chim. Acta 2000, 406, 93–104.CrossRefGoogle Scholar
  9. 9.
    Šamalikova, M.; Grandori, R. Testing the Role of Solvent Surface Tension in Protein Ionization by Electrospray. J. Mass Spectrom. 2005, 40, 503–510.CrossRefGoogle Scholar
  10. 10.
    Iavarone, A. T.; Williams, E. R. Supercharging in Electrospray Ionization: Effects on Signal and Charge. Int. J. Mass Spectrom. 2002, 219, 63–72.CrossRefGoogle Scholar
  11. 11.
    Wohlfarth, C. Surface Tension of Pure Liquids and Binary Liquid Mixtures. In Landolt-Börnstein-Group Iv Physical Chemistry Vol. XXIV; Lechner, M. D., Ed.; Springer: Berlin, 2008.Google Scholar
  12. 12.
    Pettit, G. R.; Holzapfel, C. W.; Cragg, G. M.; Herald, C. L.; Williams, P. Broad Scope Secondary Ion Mass Spectrometry. J. Nat. Prod. 1983, 46, 917–922.CrossRefGoogle Scholar
  13. 13.
    Rinehart, K. L. Fast Atom Bombardment Mass Spectrometry. Science 1982, 218, 254–260.CrossRefGoogle Scholar
  14. 14.
    Jackson, S. N.; Murray, K. K. Infrared Matrix-Assisted Laser Desorption/Ionization of Polycyclic Aromatic Hydrocarbons with a Sulfolane Matrix. Rapid Commun. Mass Spectrom. 2001, 15, 1448–1452.CrossRefGoogle Scholar
  15. 15.
    Meili, J.; Seibl, J. A New Versatile Matrix for Fast Atom Bombardment Analysis. Org. Mass Spectrom. 1984, 19, 581–582.CrossRefGoogle Scholar
  16. 16.
    Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Matrix-Assisted Ultraviolet Laser Desorption of Nonvolatile Compounds. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53–68.CrossRefGoogle Scholar
  17. 17.
    Loo, J. A.; Giordani, A. B.; Muenster, H. Observation of Intact (Heme-Bound) Myoglobin by Electrospray Ionization on a Double Focusing Mass Spectrometer. Rapid Commun. Mass Spectrom. 1993, 7, 186–189.CrossRefGoogle Scholar
  18. 18.
    Holmberg, K.; Jönsson, B.; Kronberg, B.; Lindman, B., Eds. Surfactants and Polymers in Aqueous Solutions, 2nd ed.; Wiley: Chichester, England, 2003.Google Scholar
  19. 19.
    Ogorzalek Loo, R. R.; Dales, N.; Andrews, P. C. The Effect of Detergents on Proteins Analyzed by Electrospray Ionization. In Methods in Molecular Biology: Protein and Peptide Analysis by Mass Spectrometry, Vol. LXI; Chapman, J. R., Ed.; Humana Press: Totowa, NJ, 1996; pp 141–160.Google Scholar
  20. 20.
    Barrera, N. P.; Di Bartolo, N.; Booth, P. J.; Robinson, C. V. Micelles Protect Membrane Complexes from Solution to Vacuum. Science 2008, 321, 243–250.CrossRefGoogle Scholar
  21. 21.
    Ogorzalek Loo, R. R.; Dales, N.; Andrews, P. C. Surfactant Effects on Protein Structure Examined by Electrospray Ionization Mass Spectrometry. Protein Sci. 1994, 3, 1975–1983.CrossRefGoogle Scholar
  22. 22.
    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. 2003, 75, 5739–5746.CrossRefGoogle Scholar
  23. 23.
    Sterling, H. J.; Williams, E. R. Origin of Supercharging in Electrospray Ionization of Noncovalent Complexes from Aqueous Solution. J. Am. Soc. Mass Spectrom., in press.Google Scholar

Copyright information

© American Society for Mass Spectrometry 2010

Authors and Affiliations

  1. 1.Department of Chemistry and Biochemistry, David Geffen School of MedicineUniversity of California-Los AngelesLos AngelesUSA
  2. 2.Department of Biological Chemistry, David Geffen School of MedicineUniversity of California-Los AngelesLos AngelesUSA
  3. 3.Molecular Biology InstituteUniversity of California-Los AngelesLos AngelesUSA

Personalised recommendations