Mapping Protein–Ligand Interactions in the Gas Phase Using a Functional Group Replacement Strategy. Comparison of CID and BIRD Activation Methods

  • Lu Deng
  • Elena N. Kitova
  • John S. KlassenEmail author
Research Article


Intermolecular interactions in the gaseous ions of two protein–ligand complexes, a single chain antibody (scFv) and its trisaccharide ligand (α-D-Galp-(1→2)-[α-D-Abep-(1→3)]-α-Manp-OCH3, L1) and streptavidin homotetramer (S4) and biotin (B), were investigated using a collision-induced dissociation (CID)-functional group replacement (FGR) strategy. CID was performed on protonated ions of a series of structurally related complexes based on the (scFv + L1) and (S4 + 4B) complexes, at the +10 and +13 charge states, respectively. Intermolecular interactions were identified from decreases in the collision energy required to dissociate 50 % of the reactant ion (Ec50) upon modification of protein residues or ligand functional groups. For the (scFv + L1)10+ ion, it was found that deoxygenation of L1 (at Gal C3 and C6 and Man C4 and C6) or mutation of His101 (to Ala) resulted in a decrease in Ec50 values. These results suggest that the four hydroxyl groups and His101 participate in intermolecular H-bonds. These findings agree with those obtained using the blackbody infrared radiative dissociation (BIRD)-FGR method. However, the CID-FGR method failed to reveal the relative strengths of the intermolecular interactions or establish Man C4 OH and His101 as an H-bond donor/acceptor pair. The CID-FGR method correctly identified Tyr43, but not Ser27, Trp79, and Trp120, as a stabilizing contact in the (S4 + 4B)13+ ion. In fact, mutation of Trp79 and Trp120 led to an increase in the Ec50 value. Taken together, these results suggest that the CID-FGR method, as implemented here, does not represent a reliable approach for identifying interactions in the gaseous protein–ligand complexes.

Key words

Protein–ligand complexes Collision-induced dissociation Intermolecular interactions Dissociation kinetics 



The authors are grateful for financial support provided by the Natural Sciences and Engineering Research Council of Canada and the Alberta Glycomics Centre.

Supplementary material

13361_2013_651_MOESM1_ESM.doc (1.2 mb)
ESM 1 (DOC 1274 kb)


  1. 1.
    Kitova, E., El-Hawiet, A., Schnier, P., Klassen, J.: Reliable determinations of protein–ligand interactions by direct ESI-MS measurements. Are we there yet? J. Am. Soc. Mass Spectrom 23, 431–441 (2012)CrossRefGoogle Scholar
  2. 2.
    Kitova, E.N., Seo, M., Roy, P.-N., Klassen, J.S.: Elucidating the intermolecular interactions within a desolvated protein − ligand complex. An experimental and computational study. J. Am. Chem. Soc. 130, 1214–1226 (2008)CrossRefGoogle Scholar
  3. 3.
    Price, W.D., Schnier, P.D., Jockusch, R.A., Strittmatter, E.F., Williams, E.R.: Unimolecular reaction kinetics in the high-pressure limit without collisions. J. Am. Chem. Soc. 118, 10640–10644 (1996)CrossRefGoogle Scholar
  4. 4.
    Dunbar, R.C., McMahon, T.B.: Activation of unimolecular reactions by ambient blackbody radiation. Science 279, 194–197 (1998)CrossRefGoogle Scholar
  5. 5.
    Kitova, E.N., Bundle, D.R., Klassen, J.S.: Thermal dissociation of protein − oligosaccharide complexes in the gas phase: mapping the intrinsic intermolecular interactions. J. Am. Chem. Soc. 124, 5902–5913 (2002)CrossRefGoogle Scholar
  6. 6.
    Kitova, E.N., Bundle, D.R., Klassen, J.S.: Partitioning of solvent effects and intrinsic interactions in biological recognition. Angew. Chem. Int. Ed. 43, 4183–4186 (2004)CrossRefGoogle Scholar
  7. 7.
    Liu, L., Bagal, D., Kitova, E.N., Schnier, P.D., Klassen, J.S.: Hydrophobic protein − ligand interactions preserved in the gas phase. J. Am. Chem. Soc. 131, 15980–15981 (2009)CrossRefGoogle Scholar
  8. 8.
    Liu, L., Michelsen, K., Kitova, E.N., Schnier, P.D., Klassen, J.S.: Evidence that water can reduce the kinetic stability of protein − hydrophobic ligand interactions. J. Am. Chem. Soc. 132, 17658–17660 (2010)CrossRefGoogle Scholar
  9. 9.
    Liu, L., Michelsen, K., Kitova, E.N., Schnier, P.D., Klassen, J.S.: Energetics of lipid binding in a hydrophobic protein cavity. J. Am. Chem. Soc. 134, 3054–3060 (2012)CrossRefGoogle Scholar
  10. 10.
    Deng, L., Broom, A., Kitova, E.N., Richards, M.R., Zheng, R.B., Shoemaker, G.K., Meiering, E.M., Klassen, J.S.: Kinetic stability of the streptavidin–biotin interaction enhanced in the gas phase. J. Am. Chem. Soc. 134, 16586–16596 (2012)CrossRefGoogle Scholar
  11. 11.
    McLuckey, S.A.: Principles of collisional activation in analytical mass spectrometry. J. Am. Soc. Mass Spectrom. 3, 599–614 (1992)CrossRefGoogle Scholar
  12. 12.
    Tešić, M., Wicki, J., Poon, D.K.Y., Withers, S.G., Douglas, D.J.: Gas phase noncovalent protein complexes that retain solution binding properties: binding of xylobiose inhibitors to the β-1, 4 exoglucanase from Cellulomonas fimi. J. Am. Soc. Mass Spectrom. 18, 64–73 (2007)CrossRefGoogle Scholar
  13. 13.
    Hopper, J.T.S., Oldham, N.J.: Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: the effect of ligand binding on conformational stability. J. Am. Soc. Mass Spectrom. 20, 1851–1858 (2009)CrossRefGoogle Scholar
  14. 14.
    Zdanov, A., Li, Y., Bundle, D.R., Deng, S.J., MacKenzie, C.R., Narang, S.A., Young, N.M., Cygler, M.: Structure of a single-chain antibody variable domain (Fv) fragment complexed with a carbohydrate antigen at 1.7-A resolution. Proc. Natl. Acad. Sci. U. S. A. 91, 6423–6427 (1994)CrossRefGoogle Scholar
  15. 15.
    Bundle, D.R., Baumann, H., Brisson, J.-R., Gagne, S.M., Zdanov, A., Cygler, M.: The solution structure of a trisaccharide-antibody complex: comparison of NMR measurements with a crystal structure. Biochemistry 33, 5183–5192 (1994)CrossRefGoogle Scholar
  16. 16.
    Chilkoti, A., Tan, P.H., Stayton, P.S.: Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120. Proc. Natl. Acad. Sci. U. S. A. 92, 1754–1758 (1995)CrossRefGoogle Scholar
  17. 17.
    Wang, W., Kitova, E.N., Klassen, J.S.: Nonspecific Protein − Carbohydrate Complexes Produced by Nanoelectrospray Ionization. Factors influencing their formation and stability. Anal. Chem. 77, 3060–3071 (2005)CrossRefGoogle Scholar
  18. 18.
    Sun, J., Kitova, E.N., Klassen, J.S.: Method for stabilizing protein − ligand complexes in nanoelectrospray ionization mass spectrometry. Anal. Chem. 79, 416–425 (2006)CrossRefGoogle Scholar
  19. 19.
    Hopper, J., Rawlings, A., Afonso, J., Channing, D., Layfield, R., Oldham, N.: Evidence for the preservation of native inter- and intra-molecular hydrogen bonds in the desolvated FK-binding protein·FK506 complex produced by electrospray ionization. J. Am. Soc. Mass Spectrom. 23, 1757–1767 (2012)CrossRefGoogle Scholar
  20. 20.
    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
  21. 21.
    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. Protocols 3, 1139–1152 (2008)CrossRefGoogle Scholar
  22. 22.
    Mesleh, M.F., Hunter, J.M., Shvartsburg, A.A., Schatz, G.C., Jarrold, M.F.: Structural information from ion mobility measurements: effects of the long-range potential. J. Phys. Chem. 100, 16082–16086 (1996)CrossRefGoogle Scholar
  23. 23.
    Vékey, K.: Internal energy effects in mass spectrometry. J. Mass Spectrom. 31, 445–463 (1996)CrossRefGoogle Scholar
  24. 24.
    Mayer, P.M., Poon, C.: The mechanisms of collisional activation of ions in mass spectrometry. Mass Spectrom. Rev. 28, 608–639 (2009)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2013

Authors and Affiliations

  1. 1.Department of Chemistry and Alberta Glycomics CentreUniversity of AlbertaEdmontonCanada

Personalised recommendations