Journal of The American Society for Mass Spectrometry

, Volume 28, Issue 9, pp 1863–1875 | Cite as

Insight into Signal Response of Protein Ions in Native ESI-MS from the Analysis of Model Mixtures of Covalently Linked Protein Oligomers

  • Katharina Root
  • Yves Wittwer
  • Konstantin Barylyuk
  • Ulrike Anders
  • Renato Zenobi
Research Article


Native ESI-MS is increasingly used for quantitative analysis of biomolecular interactions. In such analyses, peak intensity ratios measured in mass spectra are treated as abundance ratios of the respective molecules in solution. While signal intensities of similar-size analytes, such as a protein and its complex with a small molecule, can be directly compared, significant distortions of the peak ratio due to unequal signal response of analytes impede the application of this approach for large oligomeric biomolecular complexes. We use a model system based on concatenated maltose binding protein units (MBPn, n = 1, 2, 3) to systematically study the behavior of protein mixtures in ESI-MS. The MBP concatamers differ from each other only by their mass while the chemical composition and other properties remain identical. We used native ESI-MS to analyze model mixtures of MBP oligomers, including equimolar mixtures of two proteins, as well as binary mixtures containing different fractions of the individual components. Pronounced deviation from a linear dependence of the signal intensity with concentration was observed for all binary mixtures investigated. While equimolar mixtures showed linear signal dependence at low concentrations, distinct ion suppression was observed above 20 μM. We systematically studied factors that are most often used in the literature to explain the origin of suppression effects. Implications of this effect for quantifying protein–protein binding affinity by native ESI-MS are discussed in general and demonstrated for an example of an anti-MBP antibody with its ligand, MBP.

Graphical Abstract


Native ESIMS Response factors Concatenated protein 

Supplementary material

13361_2017_1690_MOESM1_ESM.docx (1.6 mb)
ESM 1(DOCX 1.57 MB)
13361_2017_1690_MOESM2_ESM.pdf (142 kb)
ESM 2(PDF 142 kb)
13361_2017_1690_MOESM3_ESM.pdf (149 kb)
ESM 3(PDF 148 kb)
13361_2017_1690_MOESM4_ESM.pdf (307 kb)
ESM 4(PDF 306 kb)


  1. 1.
    Robinson, C.V., Sali, A., Baumeister, W.: The molecular sociology of the cell. Nat. 450, 973–982 (2007)CrossRefGoogle Scholar
  2. 2.
    Hernandez, H., Robinson, C.V.: Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat. Protoc. 2, 715–726 (2007)CrossRefGoogle Scholar
  3. 3.
    Amr El-Hawiet, E.N.K., Klassen, J.S.: Biochemistry 51, 4244–4253 (2012)CrossRefGoogle Scholar
  4. 4.
    Jaquillard, L., Saab, F., Schoentgen, F., Cadene, M.: Improved accuracy of low affinity protein-ligand equilibrium dissociation constants directly determined by electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 23, 908–922 (2012)CrossRefGoogle Scholar
  5. 5.
    Barrera, N.P., Di Bartolo, N., Booth, P.J., Robinson, C.V.: Micelles protect membrane complexes from solution to vacuum. Science 321, 243–246 (2008)CrossRefGoogle Scholar
  6. 6.
    Laganowsky, A., Reading, E., Hopper, J.T., Robinson, C.V.: Mass spectrometry of intact membrane protein complexes. Nat. Protoc. 8, 639–651 (2013)CrossRefGoogle Scholar
  7. 7.
    Benjamin, D.R., Robinson, C.V., Hendrick, J.P., Hartl, F.U., Dobson, C.M.: Mass spectrometry of ribosomes and ribosomal subunits. Proc. Natl. Acad. Sci. U. S. A. 95, 7391–7395 (1998)CrossRefGoogle Scholar
  8. 8.
    Bernstein, S.L., Wyttenbach, T., Baumketner, A., Shea, J.E., Bitan, G., Teplow, D.B., Bowers, M.T.: Amyloid beta-protein: monomer structure and early aggregation states of A beta 42 and its Pro(19) alloform. J. Am. Chem. Soc. 127, 2075–2084 (2005)CrossRefGoogle Scholar
  9. 9.
    Bothner, B., Siuzdak, G.: Electrospray ionization of a whole virus: analyzing mass, structure, and viability. Chem. BioChem. 5, 258–260 (2004)Google Scholar
  10. 10.
    Uetrecht, C., Versluis, C., Watts, N.R., Wingfield, P.T., Steven, A.C., Heck, A.J.R.: Stability and shape of hepatitis B virus capsids in vacuo. Angew. Chem. Int. Ed. 47, 6247–6251 (2008)CrossRefGoogle Scholar
  11. 11.
    Snijder, J., Rose, R.J., Veesler, D., Johnson, J.E., Heck, A.J.R.: Studying 18 MDa virus assemblies with native mass spectrometry. Angew. Chem. Int. Ed. 52, 4020–4023 (2013)CrossRefGoogle Scholar
  12. 12.
    Heck, A.J.R., Van Den Heuvel, R.H.: Investigation of intact protein complexes by mass spectrometry. Mass Spectrom. Rev. 23, 368–389 (2004)CrossRefGoogle Scholar
  13. 13.
    Uetrecht, C., Heck, A.J.R.: Modern biomolecular mass spectrometry and its role in studying virus structure, dynamics, and assembly. Angew. Chem. Int. Ed. 50, 8248–8262 (2011)CrossRefGoogle Scholar
  14. 14.
    Fenn, J.B.: Electrospray wings for molecular elephants (Nobel lecture). Angew. Chem. Int. Ed. 42, 3871–3894 (2003)CrossRefGoogle Scholar
  15. 15.
    Benesch, J.L., Robinson, C.V.: Mass spectrometry of macromolecular assemblies: preservation and dissociation. Curr. Opin. Struct. Biol. 16, 245–251 (2006)CrossRefGoogle Scholar
  16. 16.
    van den Heuvel, R.H., Heck, A.J.R.: Native protein mass spectrometry: from intact oligomers to functional machineries. Curr. Opin. Chem. Biol. 8, 519–526 (2004)CrossRefGoogle Scholar
  17. 17.
    Chitta, R.K., Rempel, D.L., Gross, M.L.: Determination of affinity constants and response factors of the noncovalent dimer of gramicidin by electrospray ionization mass spectrometry and mathematical modeling. J. Am. Soc. Mass Spectrom. 16, 1031–1038 (2005)CrossRefGoogle Scholar
  18. 18.
    Lin, H., Kitova, E.N., Klassen, J.S.: Quantifying protein–ligand interactions by direct electrospray ionization-MS analysis: evidence of nonuniform response factors induced by high molecular weight molecules and complexes. Anal. Chem. 85, 8919–8922 (2013)CrossRefGoogle Scholar
  19. 19.
    El-Hawiet, A., Kitova, E.N., Arutyunov, D., Simpson, D.J., Szymanski, C.M., Klassen, J.S.: Quantifying ligand binding to large protein complexes using electrospray ionization mass spectrometry. Anal. Chem. 84, 3867–3870 (2012)CrossRefGoogle Scholar
  20. 20.
    Gabelica, V., Rosu, F., De Pauw, E.: A simple method to determine electrospray response factors of noncovalent complexes. Anal. Chem. 81, 6708–6715 (2009)CrossRefGoogle Scholar
  21. 21.
    Liu, J.J., Konermann, L.: Protein–protein binding affinities in solution determined by electrospray mass spectrometry. J. Am. Soc. Mass Spectrom. 22, 408–417 (2011)CrossRefGoogle Scholar
  22. 22.
    Kuprowski, M.C., Konermann, L.: Signal response of coexisting protein conformers in electrospray mass spectrometry. Anal. Chem. 79, 2499–2506 (2007)CrossRefGoogle Scholar
  23. 23.
    El-Hawiet, A., Kitova, E.N., Liu, L., Klassen, J.S.: Quantifying labile protein–ligand interactions using electrospray ionization mass spectrometry. J. Am. Soci. Mass Spectrom. 21, 1893–1899 (2010)Google Scholar
  24. 24.
    Erba, E.B., Barylyuk, K., Yang, Y., Zenobi, R.: Quantifying protein–protein interactions within noncovalent complexes using electrospray ionization mass spectrometry. Anal. Chem. 83, 9251–9259 (2011)CrossRefGoogle Scholar
  25. 25.
    Barylyuk, K., Gulbakan, B., Xie, X.S., Zenobi, R.: DNA Oligonucleotides: a model system with tunable binding strength to study monomer-dimer equilibria with electrospray ionization-mass spectrometry. Anal. Chem. 85, 11902–11912 (2013)CrossRefGoogle Scholar
  26. 26.
    Weidmann, S., Barylyuk, K., Nespovitaya, N., Madler, S., Zenobi, R.: A new, modular mass calibrant for high-mass MALDI-MS. Anal. Chem. 85, 3425–3432 (2013)CrossRefGoogle Scholar
  27. 27.
    Bush, M.F., Hall, Z., Giles, K., Hoyes, J., Robinson, C.V., Ruotolo, B.T.: Collision cross-sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal. Chem. 82, 9557–9565 (2010)CrossRefGoogle Scholar
  28. 28.
    Kebarle, P., Verkerk, U.H.: Electrospray: from ions in solution to ions in the gas phase, what we know now. Mass Spectrom. Rev. 28, 898–917 (2009)CrossRefGoogle Scholar
  29. 29.
    Loo, R.R.O., 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
  30. 30.
    Weidmann, S., Mikutis, G., Barylyuk, K., Zenobi, R.: Mass discrimination in high-mass MALDI-MS. J. Am. Soc. Mass Spectrom. 24, 1396–1404 (2013)CrossRefGoogle Scholar
  31. 31.
    Konermann, L., Ahadi, E., Rodriguez, A.D., Vahidi, S.: Unraveling the mechanism of electrospray ionization. Anal. Chem. 85, 2–9 (2013)CrossRefGoogle Scholar
  32. 32.
    de la Mora Fernandez, J.: Electrospray ionization of large multiply charged species proceeds via Dole's charged residue mechanism. Anal. Chim. Acta 406, 93–104 (2000)CrossRefGoogle Scholar
  33. 33.
    Peschke, M., Verkerk, U.H., Kebarle, P.: Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method. J. Am. Soc. Mass Spectrom. 15, 1424–1434 (2004)CrossRefGoogle Scholar
  34. 34.
    Hogan, C.J., Carroll, J.A., Rohrs, H.W., Biswas, P., Gross, M.L.: Charge carrier field emission determines the number of charges on native state proteins in electrospray ionization. J. Am. Chem. Soc. 130, 6926 (2008)CrossRefGoogle Scholar
  35. 35.
    Hogan, C.J., Carroll, J.A., Rohrs, H.W., Biswas, P., Gross, M.L.: Combined charged residue-field emission model of macromolecular electrospray ionization. Anal. Chem. 81, 369–377 (2009)CrossRefGoogle Scholar
  36. 36.
    Loscertales, I.G., de la Mora Fernandez, J.: Experiments on the kinetics of field evaporation of small ions from droplets. J. Chem. Phys. 103, 5041–5060 (1995)CrossRefGoogle Scholar
  37. 37.
    Ahadi, E., Konermann, L.: Surface charge of electrosprayed water nanodroplets: a molecular dynamics study. J. Am. Chem. Soc. 132, 11270–11277 (2010)CrossRefGoogle Scholar
  38. 38.
    Marty, M.T., Baldwin, A.J., Marklund, E.G., Hochberg, G.K.A., Benesch, J.L.P., Robinson, C.V.: Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015)CrossRefGoogle Scholar
  39. 39.
    Ruotolo, B.T., Benesch, J.L.P., Sandercock, A.M., Hyung, S.J., Robinson, C.V.: Ion mobility-mass spectrometry analysis of large protein complexes. Nat. Protoc. 3, 1139–1152 (2008)CrossRefGoogle Scholar
  40. 40.
    Larriba-Andaluz, C., Hogan, C.J.: Collision cross-section calculations for polyatomic ions considering rotating diatomic/linear gas molecules. J. Chem. Phys. 141, (2014)Google Scholar

Copyright information

© American Society for Mass Spectrometry 2017

Authors and Affiliations

  • Katharina Root
    • 1
  • Yves Wittwer
    • 1
  • Konstantin Barylyuk
    • 1
    • 2
  • Ulrike Anders
    • 1
  • Renato Zenobi
    • 1
  1. 1.Department of Chemistry and Applied BiosciencesETH ZurichZurichSwitzerland
  2. 2.Department of BiochemistryUniversity of CambridgeCambridgeUK

Personalised recommendations