Ion Mobility-Mass Spectrometry to Evaluate the Effects of Protein Modification or Small Molecule Binding on Protein Dynamics

  • Lauren J. Tomlinson
  • Claire E. EyersEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2084)


Ion mobility-mass spectrometry (IM-MS) of intact protein complexes under native conditions is a powerful tool for the analysis of protein complexes and protein–ligand interactions, permitting insight into ligand-induced changes in protein conformation. Here we describe a procedure for analyzing the effects of phosphorylation and/or inhibitor binding on protein kinase conformational flexibility using Protein Kinase A (PKA) as a model system. By calculating the protein collision cross section (CCS) before and after inhibitor binding, and additionally by performing collision-induced unfolding (CIU), we can establish the effects of protein modification or small molecule binding on protein dynamics.

Key words

Ion mobility-mass spectrometry Native mass spectrometry Collision cross section Collision-induced unfolding 


  1. 1.
    Göth M, Pagel K (2017) Ion mobility–mass spectrometry as a tool to investigate protein–ligand interactions. Anal Bioanal Chem 409:4305. Scholar
  2. 2.
    Eyers CE, Vonderach M, Ferries S, Jeacock K, Eyers PA (2018) Understanding protein–drug interactions using ion mobility–mass spectrometry. Curr Opin Chem Biol 42:167–176. Scholar
  3. 3.
    Eschweiler J, Kerr R, Rabuck-Gibbons J, Ruotolo BT (2017) Sizing up protein–ligand complexes: the rise of structural mass spectrometry approaches in the pharmaceutical sciences. Annu Rev Anal Chem 10:25–44. Scholar
  4. 4.
    Ben-Nissan G, Sharon M (2017) The application of ion-mobility mass spectrometry for structure/function investigation of protein complexes. Curr Opin Chem Biol 42:25–33. Scholar
  5. 5.
    Shuai N, Rabuck JN, Ruotolo BT (2013) Ion mobility-mass spectrometry of intact protein-ligand complexes for pharmaceutical drug discovery and development. Curr Opin Chem Biol 17(5):809–817. Scholar
  6. 6.
    Nshanian M, Lantz C, Wongkongkathep P et al (2019) Native top-down mass spectrometry and ion mobility spectrometry of the interaction of tau protein with a molecular tweezer. J Am Soc Mass Spectrom 30(1):16–23. Scholar
  7. 7.
    Byrne DP, Vonderach M, Ferries S, Brownridge PJ, Eyers CE, Eyers PA (2016) cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning Fluorimetry and ion-mobility mass spectrometry. Biochem J 473(19):3159–3175CrossRefGoogle Scholar
  8. 8.
    Wongkongkathep P, Han JY, Choi TS, Yin S, Kim HI, Loo JA (2018) Native top-down mass spectrometry and ion mobility MS for characterizing the cobalt and manganese metal binding of α-synuclein protein. J Am Soc Mass Spectrom 29(9):1870–1880. Scholar
  9. 9.
    Yuwei T, Lippens J, Netirojjanakul C, Campuzano IDG, Ruotolo B (2018) Quantitative collision-induced unfolding differentiates model antibody–drug conjugates. Protein Sci 28(3):598–608. Scholar
  10. 10.
    Rabuck-Gibbons JN, Lodge JM, Mapp AK, Ruotolo BT (2019) Collision-induced unfolding reveals unique fingerprints for remote protein interaction sites in the KIX regulation domain. J Am Soc Mass Spectrom 30(1):94–102. Scholar
  11. 11.
    Rabuck JN, Suk-Joon H, Ko KS, Fox CC, Soellner MB, Ruotolo BT (2013) Activation state-selective kinase inhibitor assay based on ion mobility-mass spectrometry. Anal Chem 85(15):6995–7002. Scholar
  12. 12.
    Saunders C, Young LM, Mahood RA, Jackson MP, Revill CH, Foster RJ, Smith AD, Ashcroft AE, Brockwell DJ, Radford SE (2016) An in vivo platform for identifying inhibitors of protein aggregation. Nat Chem Biol 12:94–101. Scholar
  13. 13.
    Hozefa A, Bate C, Williams A, Virdee J, Jeggo R, Spanswick D, Scopes DIC, Treherne JM, Mazzitelli S, Chawner R, Eyers CE, Doig AJ (2012) The N-methylated peptide SEN304 powerfully inhibits Aβ(1–42) toxicity by perturbing oligomer formation. Biochemistry 51(42):8338–8352. Scholar
  14. 14.
    Vonderach M, Byrne DP, Barran PE, Eyers PA, Eyers CE (2018) DNA binding and phosphorylation regulate the core structure of the NF-κB p50 transcription factor. J Am Soc Mass Spectrom 30(1):128–138CrossRefGoogle Scholar
  15. 15.
    Lixa C, Mujo A, de Magalhães MTQ, Almeida FCL, Lima LMTR, Pinheir AS (2018) Oligomeric transition and dynamics of RNA binding by the HuR RRM1 domain in solution. J Bimol NMR 72(3–4):179–192. Scholar
  16. 16.
    Wang Y, Park H, Lin H, Kitova EN, Klassen JS (2019) Multipronged ESI–MS approach for studying glycan-binding protein interactions with glycoproteins. Anal Chem 91(3):2140–2147. Scholar
  17. 17.
    Zhao Y, Yang JY, Thieker DF, Xu Y, Zong C, Boons GJ, Liu J, Woods RJ, Moremen KW, Amster IJ (2018) A Traveling Wave Ion Mobility Spectrometry (TWIMS) study of the Robo1-heparan sulfate interaction. J Am Soc Mass Spectrom. Scholar
  18. 18.
    Lanucara F, Holman SW, Gray CJ, Eyers CE (2014) The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nat Chem 6(4):281–294. Scholar
  19. 19.
    Gabelica V, Shvartsburg AA, Afonso C, Barran P, Benesch JLP, Bleiholder C, Bowers MT, Bilbao A, Bush MF, Larry Campbell J, Campuzano IDG, Causon T, Clowers BH, Creaser CS, De Pauw E, Far J, Fernandez-Lima F, Fjeldsted JC, Giles K, Groessl M, Hogan CJ, Hann S, Kim HI, Kurulugama RT, May JC, McLean JA, Pagel K, Richardson K, Ridgeway ME, Rosu F, Sobott F, Thalassinos K, Valentine SJ, Wyttenbach T (2017) Recommendations for reporting ion mobility Mass Spectrometry measurements. Mass Spectrom Rev 38(3):291–320CrossRefGoogle Scholar
  20. 20.
    Khajehali E, Malone DT, Glass M, Sexton PM, Christopoulos A, Leach K (2015) Biased agonism and biased allosteric modulation at the CB1 cannabinoid receptor. Mol Pharmacol 88(2):368–379. Scholar
  21. 21.
    Verkhivker GM (2017) Leveraging structural diversity and allosteric regulatory mechanisms of protein kinases in the discovery of small molecule inhibitors. Curr Med Chem 24(42):4838–4872. Scholar
  22. 22.
    Wilson LJ, Linley A, Hammond DE, Hood FE, Coulson JM, MacEwan DJ, Ross SJ, Slupsky JR, Smith PD, Eyers PA, Prior IA (2017) New perspectives, opportunities, and challenges in exploring the human protein kinome. Cancer Res 78(1):1–16. Scholar
  23. 23.
    Masterson LR, Mascioni A, Traaseth NJ, Taylor SS, Veglia G (2008) Allosteric cooperativity in protein kinase A. Proc Natl Acad Sci U S A 105:506–511CrossRefGoogle Scholar
  24. 24.
    Bush M (2019) Collision cross section database. Bush Lab.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Biochemistry, Centre for Proteome Research, Institute of Integrative BiologyUniversity of LiverpoolLiverpoolUK

Personalised recommendations