Quantification of Protein-Ligand Interactions by Laser Electrospray Mass Spectrometry

  • Jieutonne J. Archer
  • Santosh Karki
  • Fengjian Shi
  • Habiballah Sistani
  • Robert J. Levis
Research Article


Laser electrospray mass spectrometry (LEMS) measurement of the dissociation constant (Kd) for hen egg white lysozyme (HEWL) and N,N′,N″-triacetylchitotriose (NAG3) revealed an apparent Kd value of 313.2 ± 25.9 μM for the ligand titration method. Similar measurements for N,N′,N″,N″’-tetraacetylchitotetraose (NAG4) revealed an apparent Kd of 249.3 ± 13.6 μM. An electrospray ionization mass spectrometry (ESI-MS) experiment determined a Kd value of 9.8 ± 0.6 μM. In a second LEMS approach, a calibrated measurement was used to determine a Kd value of 6.8 ± 1.5 μM for NAG3. The capture efficiency of LEMS was measured to be 3.6 ± 1.8% and is defined as the fraction of LEMS sample detected after merging with the ESI plume. When the dilution is factored into the ligand titration measurement, the adjusted Kd value was 11.3 μM for NAG3 and 9.0 μM for NAG4. The calibration method for measuring Kd developed in this study can be applied to solutions containing unknown analyte concentrations.

Graphical Abstract


Femtosecond Laser Electrospray ionization Protein-ligand Dissociation constant Charge state distribution 

Supplementary material

13361_2018_1935_MOESM1_ESM.docx (1 mb)
ESM 1 (DOCX 1027 kb)


  1. 1.
    Veenstra, T.D.: Electrospray ionization mass spectrometry in the study of biomolecular non-covalent interactions. Biophys. Chem. 79, 63–79 (1999)CrossRefGoogle Scholar
  2. 2.
    Veros, C.T., Oldham, N.J.: Quantitative determination of lysozyme-ligand binding in the solution and gas phases by electrospray ionisation mass spectrometry. Rapid Commun. Mass Spectrom. 21, 3505–3510 (2007)CrossRefGoogle Scholar
  3. 3.
    Deng, G., Sanyal, G.: Applications of mass spectrometry in early stages of target based drug discovery. J. Pharm. Biomed. Anal. 40, 528–538 (2006)CrossRefGoogle Scholar
  4. 4.
    Heck, A.J., van den Heuvel, R.H.: Investigation of intact protein complexes by mass spectrometry. Mass Spectrom. Rev. 23, 368–389 (2004)CrossRefGoogle Scholar
  5. 5.
    Pramanik, B.N., Bartner, P.L., Mirza, U.A., Liu, Y.H., Ganguly, A.K.: Electrospray ionization mass spectrometry for the study of non-covalent complexes: an emerging technology. J. Mass Spectrom. 33, 911–920 (1998)CrossRefGoogle Scholar
  6. 6.
    Zhang, J., McCombie, G., Guenat, C., Knochenmuss, R.: FT-ICR mass spectrometry in the drug discovery process. Drug Discov. Today. 10, 635–642 (2005)CrossRefGoogle Scholar
  7. 7.
    Daniel, J.M., Friess, S.D., Rajagopalan, S., Wendt, S., Zenobi, R.: Quantitative determination of noncovalent binding interactions using soft ionization mass spectrometry. Int. J. Mass Spectrom. 216, 1–27 (2002)CrossRefGoogle Scholar
  8. 8.
    Jecklin, M.C., Touboul, D., Bovet, C., Wortmann, A., Zenobi, R.: Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry. J. Am. Soc. Mass Spectrom. 19, 332–343 (2008)CrossRefGoogle Scholar
  9. 9.
    Liu, L., Kitova, E.N., Klassen, J.S.: Quantifying protein-fatty acid interactions using electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 22, 310–318 (2011)CrossRefGoogle Scholar
  10. 10.
    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
  11. 11.
    De Vriendt, K., Sandra, K., Desmet, T., Nerinckx, W., Van Beeumen, J., Devreese, B.: Evaluation of automated nano-electrospray mass spectrometry in the determination of non-covalent protein–ligand complexes. Rapid Commun. Mass Spectrom. 18, 3061–3067 (2004)CrossRefGoogle Scholar
  12. 12.
    Robinson, C.V., Chung, E.W., Kragelund, B.B., Knudsen, J., Aplin, R.T., Poulsen, F.M., Dobson, C.M.: Probing the nature of noncovalent interactions by mass spectrometry. A study of protein− CoA ligand binding and assembly. J. Am. Chem. Soc. 118, 8646–8653 (1996)CrossRefGoogle Scholar
  13. 13.
    Wu, Q., Gao, J., Joseph-McCarthy, D., Sigal, G.B., Bruce, J.E., Whitesides, G.M., Smith, R.D.: Carbonic anhydrase-inhibitor binding: from solution to the gas phase. J. Am. Chem. Soc. 119, 1157–1158 (1997)CrossRefGoogle Scholar
  14. 14.
    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
  15. 15.
    Gabelica, V., Galic, N., Rosu, F., Houssier, C., De Pauw, E.: Influence of response factors on determining equilibrium association constants of non-covalent complexes by electrospray ionization mass spectrometry. J. Mass Spectrom. 38, 491–501 (2003)CrossRefGoogle Scholar
  16. 16.
    Tjernberg, A., Carnö, S., Oliv, F., Benkestock, K., Edlund, P.-O., Griffiths, W.J., Hallén, D.: Determination of dissociation constants for protein− ligand complexes by electrospray ionization mass spectrometry. Anal. Chem. 76, 4325–4331 (2004)CrossRefGoogle Scholar
  17. 17.
    Clark, S.M., Konermann, L.: Determination of ligand− protein dissociation constants by electrospray mass spectrometry-based diffusion measurements. Anal. Chem. 76, 7077–7083 (2004)CrossRefGoogle Scholar
  18. 18.
    Svobodová, J., Mathur, S., Muck, A., Letzel, T., Svatoš, A.: Microchip-ESI-MS determination of dissociation constant of the lysozyme–NAG3 complex. Electrophoresis. 31, 2680–2685 (2010)CrossRefGoogle Scholar
  19. 19.
    Flanigan, P., Levis, R.: Ambient femtosecond laser vaporization and nanosecond laser desorption electrospray ionization mass spectrometry. Annu. Rev. Anal. Chem. 7, 229–256 (2014)CrossRefGoogle Scholar
  20. 20.
    Sampson, J.S., Hawkridge, A.M., Muddiman, D.C.: Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry. J. Am. Soc. Mass Spectrom. 17, 1712–1716 (2006)CrossRefGoogle Scholar
  21. 21.
    Huang, M.-Z., Hsu, H.-J., Lee, J.-Y., Jeng, J., Shiea, J.: Direct protein detection from biological media through electrospray-assisted laser desorption ionization/mass spectrometry. J. Proteome Res. 5, 1107–1116 (2006)CrossRefGoogle Scholar
  22. 22.
    Brady, J.J., Judge, E.J., Levis, R.J.: Mass spectrometry of intact neutral macromolecules using intense non-resonant femtosecond laser vaporization with electrospray post-ionization. Rapid Commun. Mass Spectrom. 23, 3151–3157 (2009)CrossRefGoogle Scholar
  23. 23.
    Karki, S., Flanigan, P.M., Perez, J.J., Archer, J.J., Levis, R.J.: Increasing protein charge state when using laser electrospray mass spectrometry. J. Am. Soc. Mass Spectrom. 26, 706–715 (2015)CrossRefGoogle Scholar
  24. 24.
    Karki, S., Sistani, H., Archer, J.J., Shi, F., Levis, R.J.: Isolating protein charge state reduction in electrospray droplets using femtosecond laser vaporization. J. Am. Soc. Mass Spectrom. 28, 470–478 (2017)CrossRefGoogle Scholar
  25. 25.
    Shi, F., Flanigan IV, P.M., Archer, J.J., Levis, R.J.: Ambient molecular analysis of biological tissue using low-energy, femtosecond laser vaporization and nanospray postionization mass spectrometry. J. Am. Soc. Mass Spectrom. 27, 542–551 (2016)CrossRefGoogle Scholar
  26. 26.
    Shiea, J., Huang, M.Z., HSu, H.J., Lee, C.Y., Yuan, C.H., Beech, I., Sunner, J.: Electrospray-assisted laser desorption/ionization mass spectrometry for direct ambient analysis of solids. Rapid Commun. Mass Spectrom. 19, 3701–3704 (2005)CrossRefGoogle Scholar
  27. 27.
    Perez, J.J., Flanigan IV, P.M., Karki, S., Levis, R.J.: Laser electrospray mass spectrometry minimizes ion suppression facilitating quantitative mass spectral response for multicomponent mixtures of proteins. Anal. Chem. 85, 6667–6673 (2013)CrossRefGoogle Scholar
  28. 28.
    Nemes, P., Barton, A.A., Li, Y., Vertes, A.: Ambient molecular imaging and depth profiling of live tissue by infrared laser ablation electrospray ionization mass spectrometry. Anal. Chem. 80, 4575–4582 (2008)CrossRefGoogle Scholar
  29. 29.
    Nemes, P., Vertes, A.: Laser ablation electrospray ionization for atmospheric pressure, in vivo, and imaging mass spectrometry. Anal. Chem. 79, 8098–8106 (2007)CrossRefGoogle Scholar
  30. 30.
    Nemes, P., Vertes, A.: Atmospheric-pressure molecular imaging of biological tissues and biofilms by LAESI mass spectrometry. J. Vis. Exp. 43, e2097 (2010)Google Scholar
  31. 31.
    Robichaud, G., Barry, J.A., Muddiman, D.C.: IR-MALDESI mass spectrometry imaging of biological tissue sections using ice as a matrix. J. Am. Soc. Mass Spectrom. 25, 319–328 (2014)CrossRefGoogle Scholar
  32. 32.
    Shi, F., Archer, J.J., Levis, R.J.: Nonresonant, femtosecond laser vaporization and electrospray post-ionization mass spectrometry as a tool for biological tissue imaging. Methods. 104, 79–85 (2016)CrossRefGoogle Scholar
  33. 33.
    Li, H., Ha, E., Donaldson, R.P., Jeremic, A.M., Vertes, A.: Rapid assessment of human amylin aggregation and its inhibition by copper (II) ions by laser ablation electrospray ionization mass spectrometry with ion mobility separation. Anal. Chem. 87, 9829–9837 (2015)CrossRefGoogle Scholar
  34. 34.
    Lee, E.C., Ha, E., Singh, S., Legesse, L., Ahmad, S., Karnaukhova, E., Donaldson, R.P., Jeremic, A.M.: Copper (II)–human amylin complex protects pancreatic cells from amylin toxicity. Phys. Chem. Chem. Phys. 15, 12558–12571 (2013)CrossRefGoogle Scholar
  35. 35.
    Brady, J.J., Judge, E.J., Levis, R.J.: Nonresonant femtosecond laser vaporization of aqueous protein preserves folded structure. Proc. Natl. Acad. Sci. U. S. A. 108, 12217–12222 (2011)CrossRefGoogle Scholar
  36. 36.
    Akhterov, M.V., Choi, Y., Olsen, T.J., Sims, P.C., Iftikhar, M., Gul, O.T., Corso, B.L., Weiss, G.A., Collins, P.G.: Observing Lysozyme’s closing and opening motions by high-resolution single-molecule enzymology. ACS Chem. Biol. 10, 1495–1501 (2015)CrossRefGoogle Scholar
  37. 37.
    Flanigan, P.M., Shi, F., Archer, J.J., Levis, R.J.: Internal energy deposition for low energy, femtosecond laser vaporization and nanospray post-ionization mass spectrometry using thermometer ions. J. Am. Soc. Mass Spectrom. 26, 716–724 (2015)CrossRefGoogle Scholar
  38. 38.
    Flanigan, P.M., Shi, F., Perez, J.J., Karki, S., Pfeiffer, C., Schafmeister, C., Levis, R.J.: Determination of internal energy distributions of laser electrospray mass spectrometry using thermometer ions and other biomolecules. J. Am. Soc. Mass Spectrom. 25, 1572–1582 (2014)CrossRefGoogle Scholar
  39. 39.
    Brady, J.J., Judge, E.J., Levis, R.J.: Analysis of amphiphilic lipids and hydrophobic proteins using nonresonant femtosecond laser vaporization with electrospray post-ionization. J. Am. Soc. Mass Spectrom. 22, 762–772 (2011)CrossRefGoogle Scholar
  40. 40.
    Shi, F., Flanigan IV, P.M., Archer, J.J., Levis, R.J.: Direct analysis of intact biological macromolecules by low-energy, fiber-based femtosecond laser vaporization at 1042 nm wavelength with nanospray postionization mass spectrometry. Anal. Chem. 87, 3187–3194 (2015)CrossRefGoogle Scholar
  41. 41.
    Sistani, H., Karki, S., Archer, J.J., Shi, F., Levis, R.J.: Assessment of reproducibility of laser electrospray mass spectrometry using electrospray deposition of analyte. J. Am. Soc. Mass Spectrom. 28, 880–886 (2017)CrossRefGoogle Scholar
  42. 42.
    Cubrilovic, D., Zenobi, R.: Influence of dimehylsulfoxide on protein–ligand binding affinities. Anal. Chem. 85, 2724–2730 (2013)CrossRefGoogle Scholar
  43. 43.
    Imoto, T., Johnson, L., North, A., Phillips, D., Rupley, J.: 21 vertebrate lysozymes. The enzymes. 7, 665–868 (1972)CrossRefGoogle Scholar
  44. 44.
    Schindler, M., Assaf, Y., Sharon, N., Chipman, D.M.: Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes. Biochemist. 16, 423–431 (1977)CrossRefGoogle Scholar
  45. 45.
    Yao, Y., Shams-Ud-Doha, K., Daneshfar, R., Kitova, E.N., Klassen, J.S.: Quantifying protein-carbohydrate interactions using liquid sample desorption electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 26, 98–106 (2015)CrossRefGoogle Scholar
  46. 46.
    Liu, P., Zhang, J., Ferguson, C.N., Chen, H., Loo, J.A.: Measuring protein–ligand interactions using liquid sample desorption electrospray ionization mass spectrometry. Anal. Chem. 85, 11966–11972 (2013)CrossRefGoogle Scholar
  47. 47.
    Judge, E.J., Brady, J.J., Dalton, D., Levis, R.J.: Analysis of pharmaceutical compounds from glass, fabric, steel, and wood surfaces at atmospheric pressure using spatially resolved, nonresonant femtosecond laser vaporization electrospray mass spectrometry. Anal. Chem. 82, 3231–3238 (2010)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Jieutonne J. Archer
    • 1
  • Santosh Karki
    • 1
  • Fengjian Shi
    • 1
  • Habiballah Sistani
    • 1
  • Robert J. Levis
    • 1
  1. 1.Department of Chemistry and Center for Advanced Photonics ResearchTemple UniversityPhiladelphiaUSA

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