Advertisement

Protein-Ligand Interaction by Ligand Titration, Fast Photochemical Oxidation of Proteins and Mass Spectrometry: LITPOMS

  • Xiaoran Roger Liu
  • Mengru Mira Zhang
  • Don L. Rempel
  • Michael L. GrossEmail author
Short Communication

Abstract

We report a novel method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) to characterize protein-ligand binding stoichiometry, binding sites, and site-specific binding constants. The system used to test the method is melittin–calmodulin, in which the peptide melittin binds to calcium-bound calmodulin. Global-level measurements reveal the binding stoichiometry of 1:1 whereas peptide-level data coupled with fitting reveal the binding sites and the site-specific binding affinity. Moreover, we extended the analysis to the residue level and identified six critical binding residues. The results show that melittin binds to the N-terminal, central linker, and C-terminal regions of holo-calmodulin with an affinity of 4.6 nM, in agreement with results of previous studies. LITPOMS, for the first time, brings high residue-level resolution to affinity measurements, providing simultaneously qualitative and quantitative understanding of protein-ligand binding. The approach can be expanded to other binding systems without tagging the protein to give high spatial resolution.

Graphical Abstract

Keywords

LITPOMS Fast photochemical oxidation of proteins (FPOP) Ligand titration Binding affinity Melittin Calmodulin Site-specific binding 

Notes

Acknowledgements

This work is financially supported by National Institute of Health NIGMS Grant 5P41GM103422 and 1S10OD016298-01A1 (to M.L.G.). Authors are grateful to Dr. Jagat Adhikari for helpful discussions and to Protein Metrics for software support.

Supplementary material

13361_2018_2076_MOESM1_ESM.docx (1 mb)
ESM 1 (DOCX 1035 kb)

References

  1. 1.
    Schellman, J.A.: Macromolecular binding. Biopolymers. 14, 999–1018 (1975)CrossRefGoogle Scholar
  2. 2.
    Willams, M.A., Daviter, T.: Protein-Ligand Interactions, Methods and Applications, 2nd edn. Humana Press, New York (2013)CrossRefGoogle Scholar
  3. 3.
    Rossi, A.M., Taylor, C.W.: Analysis of protein-ligand interactions by fluorescence polarization. Nat. Protoc. 6, 365–387 (2011)CrossRefGoogle Scholar
  4. 4.
    Yan, Y.; Marriott, G.: Analysis of protein interactions using fluorescence technologies Curr. Opin. Chem. Biol. 7, 635–640 (2003)Google Scholar
  5. 5.
    Meyer, B., Peters, T.: NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew.Chem. Int. Ed. 42, 864–890 (2003)CrossRefGoogle Scholar
  6. 6.
    Johnsson, B., Lofas, S., Lindquist, G.: Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal. Biochem. 198, 268–277 (1991)CrossRefGoogle Scholar
  7. 7.
    Willets, K. A.; Van Duyne, R. P.: Localized surface plasmon resonance spectroscopy and sensing. Annu. Rev. Phys. Chem. 58, 267–297 (2007)Google Scholar
  8. 8.
    Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L-N.: Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137 (1989)Google Scholar
  9. 9.
    Jorgensen, T.J.D., Roepstorff, P., Heck, A.J.R.: Direct determination of solution binding constants for noncovalent complexes between bacterial cell wall peptide analogues and vancomycin group antibiotics by electrospray ionization mass spectrometry. Anal. Chem. 70, 4427–4432 (1998)CrossRefGoogle Scholar
  10. 10.
    Wang, W., Kitova, E.N., Klassen, J.S.: Influence of solution and gas phase processes on protein-carbohydrate binding affinities determined by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 75, 4945–4955 (2003)CrossRefGoogle Scholar
  11. 11.
    Daneshfar, R., Kitova, E.N., Klassen, J.S.: Determination of protein-ligand association thermochemistry using variable-temperature nanoelectrospray mass spectrometry. J. Am. Chem. Soc. 126, 4786–4787 (2004)CrossRefGoogle Scholar
  12. 12.
    Gulbakan, B., Barylyuk, K., Schneider, P., Pillong, M., Schneider, G., Zenobi, R.: Native electrospray ionization mass spectrometry reveals multiple facets of aptamer-ligand interactions: from mechanism to binding constants. J. Am. Chem. Soc. 140, 7486–7497 (2018)CrossRefGoogle Scholar
  13. 13.
    Powell, K.D., Ghaemmaghami, S., Wang, M.Z., Ma, L., Oas, T.G., Fitzgerald, M.C.: A general mass spectrometry-bases assay for quantitation of protein-ligand binding interactions in solution. J. Am. Chem. Soc. 124, 10256–10257 (2002)CrossRefGoogle Scholar
  14. 14.
    Zhu, M.M., Rempel, D.L., Du, Z., Gross, M.L.: Quantification of protein-ligand interactions by mass spectrometry, titration and H/D exchange: PLIMSTEX. J. Am. Chem. Soc. 125, 5252–5253 (2003)CrossRefGoogle Scholar
  15. 15.
    Zhu, M.M., Rempel, D.L., Gross, M.L.: Modeling data from titration, amide H/D exchange, and mass spectrometry to obtain protein-ligand binding constants. J. Am. Soc. Mass Spectrom. 15, 388–397 (2004)CrossRefGoogle Scholar
  16. 16.
    Sperry, J.B., Shi, X., Rempel, D.L., Nishimura, Y., Akashi, S., Gross, M.L.: A mass spectrometric approach to the study of DNA-binding proteins: interaction of human TRF2 with telomeric DNA. Biochemistry. 47, 1797–1807 (2008)CrossRefGoogle Scholar
  17. 17.
    Sperry, J.B., Huang, R.Y., Zhu, M.M., Rempel, D.L., Gross, M.L.: Hydrophobic peptides affect binding of calmodulin and Ca2+ as explored by H/D amide exchange and mass spectrometry. Int. J. Mass Spectrom. 302, 85–92 (2011)CrossRefGoogle Scholar
  18. 18.
    Huang, R.Y.-C., Rempel, D.L., Gross, M.L.: HD exchange and PLIMSTEX determine the affinities and order of binding Ca2+ with Troponin C. Biochemistry. 50, 5426–5435 (2011)CrossRefGoogle Scholar
  19. 19.
    Wang, H., Rempel, D.L., Giblin, D., Frieden, C., Gross, M.L.: Peptide-level interactions between proteins and small-molecule drug candidates by two hydrogen-deuterium exchange MS-based methods: the example of apolipoprotein E3. Anal. Chem. 89, 10687–10695 (2017)CrossRefGoogle Scholar
  20. 20.
    Aebersold, R., Goodlett, D.R.: Mass spectrometry in proteomics. Chem. Rev. 101, 269–296 (2001)CrossRefGoogle Scholar
  21. 21.
    Aebersold, R., Mann, M.: Mass spectrometry-based proteomics. Nature. 422, 198–207 (2003)CrossRefGoogle Scholar
  22. 22.
    Weis, D. D. Hydrogen Exchange Mass Spectrometry of Proteins, Fundamentals, Methods, and Applications; Wiley: Chichester, 2016Google Scholar
  23. 23.
    Xu, G., Chance, M.R.: Hydroxyl radical-mediated modification of proteins as probes for structural proteomics. Chem. Rev. 107, 3514–3543 (2007)CrossRefGoogle Scholar
  24. 24.
    Hambly, D.M., Gross, M.L.: Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale. J. Am. Soc. Mass Spectrom. 16, 2057–2063 (2005)CrossRefGoogle Scholar
  25. 25.
    Li, K.S., Shi, L., Gross, M.L.: Mass spectrometry-based fast photochemical oxidation of proteins (FPOP) for higher order structure characterization. Acc. Chem. Res. 51, 736–744 (2018)CrossRefGoogle Scholar
  26. 26.
    Chen, J., Rempel, D.L., Gau, B.C., Gross, M.L.: Fast photochemical oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino-acid level. J. Am. Chem. Soc. 134, 18724–18731 (2012)CrossRefGoogle Scholar
  27. 27.
    Gau, B.C., Sharp, J.S., Rempel, D.L., Gross, M.L.: Fast photochemical oxidation of proteins footprints faster than protein unfolding. Anal. Chem. 81, 6563–6571 (2009)CrossRefGoogle Scholar
  28. 28.
    Zhang, B., Cheng, M., Rempel, D., Gross, M.L.: Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural. Biol. Methods. 144, 94–103 (2018)Google Scholar
  29. 29.
    Zhang, H., Gau, B.C., Jones, L.M., Vidavsky, I., Gross, M.L.: Fast photochemical oxidation of protein for comparing structures of protein-ligand complexes: the calmodulin-peptide model system. Anal. Chem. 83, 311–318 (2011)CrossRefGoogle Scholar
  30. 30.
    Comte, M., Maulet, Y., Cox, J.A.: Ca2+-dependent high-affinity complex formation between calmodulin and melittin. Biochem. J. 209, 269–272 (1983)CrossRefGoogle Scholar
  31. 31.
    Scaloni, A., Miraglia, N., Orru, S., Amodeo, P., Motta, A., Marino, G., Pucci, P.: Topology of the calmodulin-melittin complex. J. Mol. Biol. 277, 945–958 (1998)CrossRefGoogle Scholar
  32. 32.
    Schulz, D.M., Ihling, C., Clore, G.M., Sinz, A.: Mapping the topology and determination of a low-resolution three-dimensional structure of the calmodulin-melittin complex by chemical crosslinking and high-resolution FTICRMS: direct demonstration of multiple binding modes. Biochemistry. 43, 4703–4715 (2004)CrossRefGoogle Scholar
  33. 33.
    Wong, J.W.H., Maleknia, S.D., Downard, K.M.: Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 16, 225–233 (2005)CrossRefGoogle Scholar
  34. 34.
    Cheng, M., Zhang, B., Cui, W., Gross, M.L.: Liser-initiated trifluoromethylation of peptides and proteins: application to mass-spectrometry-based protein footprinting. Angew. Chem. Int. Ed. 56, 14007–14010 (2017)CrossRefGoogle Scholar
  35. 35.
    Zhang, B., Rempel, D.L., Gross, M.L.: Protein footprinting by carbenes on a fast photochemical oxidation of proteins (FPOP) platform. J. Am. Soc. Mass Spectrom. 27, 552–555 (2016)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Xiaoran Roger Liu
    • 1
  • Mengru Mira Zhang
    • 1
  • Don L. Rempel
    • 1
  • Michael L. Gross
    • 1
    Email author
  1. 1.Department of ChemistryWashington University in St. LouisSt. LouisUSA

Personalised recommendations