Native Mass Spectrometry for the Characterization of Structure and Interactions of Membrane Proteins

  • Jeroen F. van Dyck
  • Albert Konijnenberg
  • Frank Sobott
Part of the Methods in Molecular Biology book series (MIMB, volume 1635)


Over the past years, native mass spectrometry and ion mobility have grown into techniques that are widely applicable to the study of aspects of protein structure. More recently, it has become apparent that this approach provides a very promising avenue for the investigation of integral membrane proteins in lipid or detergent environments.

In this chapter, we discuss applications of native mass spectrometry and ion mobility in membrane protein research—what is important to take into consideration when working with membrane proteins, and what the requirements are for sample preparation for native mass spectrometry. Furthermore, we will discuss the types of information provided by the measurements, including the oligomeric state, subunit composition and stoichiometry, interactions with detergents or lipids, conformational transitions, and the binding and structural effect of ligands and drugs.

Key words

Native mass spectrometry Ion mobility Membrane proteins Detergent micelles Lipids 


  1. 1.
    Hopper JT, Robinson CV (2014) Mass spectrometry quantifies protein interactions – from molecular chaperones to membrane porins. Angew Chem Int Ed Engl 53(51):14002–14015CrossRefPubMedGoogle Scholar
  2. 2.
    Konijnenberg A et al (2015) Extending native mass spectrometry approaches to integral membrane proteins. Biol Chem 396(9–10):991–1002PubMedGoogle Scholar
  3. 3.
    Landreh M, Robinson CV (2015) A new window into the molecular physiology of membrane proteins. J Physiol 593(2):355–362CrossRefPubMedGoogle Scholar
  4. 4.
    Beveridge R et al (2013) Mass spectrometry methods for intrinsically disordered proteins. Analyst 138(1):32–42CrossRefPubMedGoogle Scholar
  5. 5.
    van den Heuvel RH, Heck AJ (2004) Native protein mass spectrometry: from intact oligomers to functional machineries. Curr Opin Chem Biol 8(5):519–526CrossRefPubMedGoogle Scholar
  6. 6.
    Sharon M, Robinson CV (2007) The role of mass spectrometry in structure elucidation of dynamic protein complexes. Annu Rev Biochem 76:167–193CrossRefPubMedGoogle Scholar
  7. 7.
    Konijnenberg A, Butterer A, Sobott F (2013) Native ion mobility-mass spectrometry and related methods in structural biology. Biochim Biophys Acta 1834(6):1239–1256CrossRefPubMedGoogle Scholar
  8. 8.
    Marcoux J, Robinson CV (2013) Twenty years of gas phase structural biology. Structure 21(9):1541–1550CrossRefPubMedGoogle Scholar
  9. 9.
    Hyung SJ, Ruotolo BT (2012) Integrating mass spectrometry of intact protein complexes into structural proteomics. Proteomics 12(10):1547–1564CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lorenzen K, van Duijn E (2010) Native mass spectrometry as a tool in structural biology. Curr Protoc Protein Sci Chapter 17:Unit17.12Google Scholar
  11. 11.
    Tsai YC et al (2012) Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes. J Biol Chem 287(24):20471–20481CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Marcoux J et al (2013) Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc Natl Acad Sci U S A 110(24):9704–9709CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Woods LA, Radford SE, Ashcroft AE (2013) Advances in ion mobility spectrometry-mass spectrometry reveal key insights into amyloid assembly. Biochim Biophys Acta 1834(6):1257–1268CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Knapman TW et al (2013) Ion mobility spectrometry-mass spectrometry of intrinsically unfolded proteins: trying to put order into disorder. Curr Anal Chem 9(2):181–191PubMedPubMedCentralGoogle Scholar
  15. 15.
    Jurneczko E et al (2012) Intrinsic disorder in proteins: a challenge for (un)structural biology met by ion mobility-mass spectrometry. Biochem Soc Trans 40(5):1021–1026CrossRefPubMedGoogle Scholar
  16. 16.
    Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5(12):993–996CrossRefPubMedGoogle Scholar
  17. 17.
    Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666(1–2):105–117CrossRefPubMedGoogle Scholar
  18. 18.
    Kovari LC, Momany C, Rossmann MG (1995) The use of antibody fragments for crystallization and structure determinations. Structure 3(12):1291–1293CrossRefPubMedGoogle Scholar
  19. 19.
    Steyaert J, Kobilka BK (2011) Nanobody stabilization of G protein-coupled receptor conformational states. Curr Opin Struct Biol 21(4):567–572CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Morgner N et al (2007) A novel approach to analyze membrane proteins by laser mass spectrometry: from protein subunits to the integral complex. J Am Soc Mass Spectrom 18(8):1429–1438CrossRefPubMedGoogle Scholar
  21. 21.
    Fenn JB et al (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246(4926):64–71CrossRefPubMedGoogle Scholar
  22. 22.
    Meng CK, Fenn JB (1990) Analyzing organic molecules with electrospray mass spectrometry. Am Biotechnol Lab 8(4):54–60PubMedGoogle Scholar
  23. 23.
    Mora JF et al (2000) Electrochemical processes in electrospray ionization mass spectrometry. J Mass Spectrom 35(8):939–952CrossRefPubMedGoogle Scholar
  24. 24.
    Testa L, Brocca S, Grandori R (2011) Charge-surface correlation in electrospray ionization of folded and unfolded proteins. Anal Chem 83(17):6459–6463CrossRefPubMedGoogle Scholar
  25. 25.
    D’Urzo A et al (2015) Molecular basis for structural heterogeneity of an intrinsically disordered protein bound to a partner by combined ESI-IM-MS and modeling. J Am Soc Mass Spectrom 26(3):472–481CrossRefPubMedGoogle Scholar
  26. 26.
    Barylyuk K et al (2011) What happens to hydrophobic interactions during transfer from the solution to the gas phase? The case of electrospray-based soft ionization methods. J Am Soc Mass Spectrom 22(7):1167–1177CrossRefPubMedGoogle Scholar
  27. 27.
    Nemeth-Cawley JF, Rouse JC (2002) Identification and sequencing analysis of intact proteins via collision-induced dissociation and quadrupole time-of-flight mass spectrometry. J Mass Spectrom 37(3):270–282CrossRefPubMedGoogle Scholar
  28. 28.
    Lemoine J et al (1993) Collision-induced dissociation of alkali metal cationized and permethylated oligosaccharides: influence of the collision energy and of the collision gas for the assignment of linkage position. J Am Soc Mass Spectrom 4(3):197–203CrossRefPubMedGoogle Scholar
  29. 29.
    Benesch JL, Robinson CV (2006) Mass spectrometry of macromolecular assemblies: preservation and dissociation. Curr Opin Struct Biol 16(2):245–251CrossRefPubMedGoogle Scholar
  30. 30.
    Pagel K et al (2010) Alternate dissociation pathways identified in charge-reduced protein complex ions. Anal Chem 82(12):5363–5372CrossRefPubMedGoogle Scholar
  31. 31.
    Jurchen JC, Williams ER (2003) Origin of asymmetric charge partitioning in the dissociation of gas-phase protein homodimers. J Am Chem Soc 125(9):2817–2826CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Benesch JL et al (2006) Tandem mass spectrometry reveals the quaternary organization of macromolecular assemblies. Chem Biol 13(6):597–605CrossRefPubMedGoogle Scholar
  33. 33.
    Lanucara F et al (2014) The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nat Chem 6(4):281–294CrossRefPubMedGoogle Scholar
  34. 34.
    Smith DP et al (2009) Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur J Mass Spectrom (Chichester, Eng) 15(2):113–130CrossRefGoogle Scholar
  35. 35.
    Bush MF et al (2010) Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal Chem 82(22):9557–9565CrossRefPubMedGoogle Scholar
  36. 36.
    Zhong Y, Han L, Ruotolo BT (2014) Collisional and Coulombic unfolding of gas-phase proteins: high correlation to their domain structures in solution. Angew Chem Int Ed Engl 53(35):9209–9212CrossRefPubMedGoogle Scholar
  37. 37.
    Hopper JT, Oldham NJ (2009) 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(10):1851–1858CrossRefPubMedGoogle Scholar
  38. 38.
    Laganowsky A et al (2014) Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510(7503):172–175CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Marty MT et al (2016) Probing the lipid Annular Belt by gas-phase dissociation of membrane proteins in Nanodiscs. Angew Chem Int Ed Engl 55(2):550–554CrossRefPubMedGoogle Scholar
  40. 40.
    Pan P, McLuckey SA (2003) The effect of small cations on the positive electrospray responses of proteins at low pH. Anal Chem 75(20):5468–5474CrossRefPubMedGoogle Scholar
  41. 41.
    Iavarone AT, Udekwu OA, Williams ER (2004) Buffer loading for counteracting metal salt-induced signal suppression in electrospray ionization. Anal Chem 76(14):3944–3950CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Hernandez H, Robinson CV (2007) Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat Protoc 2(3):715–726CrossRefPubMedGoogle Scholar
  43. 43.
    Hopper JT et al (2013) Detergent-free mass spectrometry of membrane protein complexes. Nat Methods 10(12):1206–1208CrossRefPubMedGoogle Scholar
  44. 44.
    Kalipatnapu S, Chattopadhyay A (2005) Membrane protein solubilization: recent advances and challenges in solubilization of serotonin1A receptors. IUBMB Life 57(7):505–512CrossRefPubMedGoogle Scholar
  45. 45.
    Laganowsky A et al (2013) Mass spectrometry of intact membrane protein complexes. Nat Protoc 8(4):639–651CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Borysik AJ, Hewitt DJ, Robinson CV (2013) Detergent release prolongs the lifetime of native-like membrane protein conformations in the gas-phase. J Am Chem Soc 135(16):6078–6083CrossRefPubMedGoogle Scholar
  47. 47.
    Dorwart MR et al (2010) S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: implications for detergent-solubilized membrane proteins. PLoS Biol 8(12):e1000555CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Konijnenberg A et al (2014) Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry. Proc Natl Acad Sci U S A 111(48):17170–17175CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Reading E et al (2015) The role of the detergent micelle in preserving the structure of membrane proteins in the gas phase. Angew Chem Int Ed Engl 54(15):4577–4581CrossRefPubMedGoogle Scholar
  50. 50.
    Reading E et al (2015) The effect of detergent, temperature, and lipid on the Oligomeric state of MscL constructs: insights from mass spectrometry. Chem Biol 22(5):593–603CrossRefPubMedGoogle Scholar
  51. 51.
    Wang SC et al (2010) Ion mobility mass spectrometry of two tetrameric membrane protein complexes reveals compact structures and differences in stability and packing. J Am Chem Soc 132(44):15468–15470CrossRefPubMedGoogle Scholar
  52. 52.
    Lee AG (2004) How lipids affect the activities of integral membrane proteins. Biochim Biophys Acta 1666(1–2):62–87CrossRefPubMedGoogle Scholar
  53. 53.
    Marty MT et al (2012) Native mass spectrometry characterization of intact nanodisc lipoprotein complexes. Anal Chem 84(21):8957–8960CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Schuler MA, Denisov IG, Sligar SG (2013) Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol Biol 974:415–433CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Bayburt TH, Sligar SG (2010) Membrane protein assembly into nanodiscs. FEBS Lett 584(9):1721–1727CrossRefPubMedGoogle Scholar
  56. 56.
    Ritchie TK et al (2009) Chapter 11 - reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol 464:211–231CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Sanders CR, Prosser RS (1998) Bicelles: a model membrane system for all seasons? Structure 6(10):1227–1234CrossRefPubMedGoogle Scholar
  58. 58.
    Vold RR, Prosser RS, Deese AJ (1997) Isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution NMR studies of polypeptides. J Biomol NMR 9(3):329–335CrossRefPubMedGoogle Scholar
  59. 59.
    Knowles TJ et al (2009) Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J Am Chem Soc 131(22):7484–7485CrossRefPubMedGoogle Scholar
  60. 60.
    Calabrese AN et al (2015) Amphipols outperform dodecylmaltoside micelles in stabilizing membrane protein structure in the gas phase. Anal Chem 87(2):1118–1126CrossRefPubMedGoogle Scholar
  61. 61.
    Gohon Y et al (2004) Partial specific volume and solvent interactions of amphipol A8-35. Anal Biochem 334(2):318–334CrossRefPubMedGoogle Scholar
  62. 62.
    Wilm M, Mann M (1996) Analytical properties of the nanoelectrospray ion source. Anal Chem 68(1):1–8CrossRefPubMedGoogle Scholar
  63. 63.
    Sobott F et al (2002) A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal Chem 74(6):1402–1407CrossRefPubMedGoogle Scholar
  64. 64.
    Landreh M et al (2015) Controlling release, unfolding and dissociation of membrane protein complexes in the gas phase through collisional cooling. Chem Commun (Camb) 51(85):15582–15584CrossRefGoogle Scholar
  65. 65.
    Chernushevich IV, Thomson BA (2004) Collisional cooling of large ions in electrospray mass spectrometry. Anal Chem 76(6):1754–1760CrossRefPubMedGoogle Scholar
  66. 66.
    Ilag LL et al (2004) Drug binding revealed by tandem mass spectrometry of a protein-micelle complex. J Am Chem Soc 126(44):14362–14363CrossRefPubMedGoogle Scholar
  67. 67.
    Lossl P, Snijder J, Heck AJ (2014) Boundaries of mass resolution in native mass spectrometry. J Am Soc Mass Spectrom 25(6):906–917CrossRefPubMedGoogle Scholar
  68. 68.
    van den Heuvel RH et al (2006) Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. Anal Chem 78(21):7473–7483CrossRefPubMedGoogle Scholar
  69. 69.
    Michaelevski I, Kirshenbaum N, Sharon M (2010) T-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis. J Vis Exp (41):e1985Google Scholar
  70. 70.
    Dyachenko A et al (2015) Tandem native mass-spectrometry on antibody-drug conjugates and Submillion da antibody-antigen protein assemblies on an Orbitrap EMR equipped with a high-mass Quadrupole mass selector. Anal Chem 87(12):6095–6102CrossRefPubMedGoogle Scholar
  71. 71.
    Rose RJ et al (2012) High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat Methods 9(11):1084–1086CrossRefPubMedGoogle Scholar
  72. 72.
    Barrera NP, Zhou M, Robinson CV (2013) The role of lipids in defining membrane protein interactions: insights from mass spectrometry. Trends Cell Biol 23(1):1–8CrossRefPubMedGoogle Scholar
  73. 73.
    Grandori R (2003) Origin of the conformation dependence of protein charge-state distributions in electrospray ionization mass spectrometry. J Mass Spectrom 38(1):11–15CrossRefPubMedGoogle Scholar
  74. 74.
    Samalikova M, Grandori R (2003) Role of opposite charges in protein electrospray ionization mass spectrometry. J Mass Spectrom 38(9):941–947CrossRefPubMedGoogle Scholar
  75. 75.
    Mehmood S et al (2014) Charge reduction stabilizes intact membrane protein complexes for mass spectrometry. J Am Chem Soc 136(49):17010–17012CrossRefPubMedPubMedCentralGoogle Scholar
  76. 76.
    Steinberg MZ et al (2007) The dynamics of water evaporation from partially solvated cytochrome c in the gas phase. Phys Chem Chem Phys 9(33):4690–4697CrossRefPubMedGoogle Scholar
  77. 77.
    Patriksson A, Marklund E, van der Spoel D (2007) Protein structures under electrospray conditions. Biochemistry 46(4):933–945CrossRefPubMedGoogle Scholar
  78. 78.
    Barrera NP et al (2009) Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nat Methods 6(8):585–587CrossRefPubMedPubMedCentralGoogle Scholar
  79. 79.
    Politis A et al (2014) A mass spectrometry-based hybrid method for structural modeling of protein complexes. Nat Methods 11(4):403–406CrossRefPubMedPubMedCentralGoogle Scholar
  80. 80.
    Zhou M et al (2011) Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334(6054):380–385CrossRefPubMedPubMedCentralGoogle Scholar
  81. 81.
    Hall Z, Politis A, Robinson CV (2012) Structural modeling of heteromeric protein complexes from disassembly pathways and ion mobility-mass spectrometry. Structure 20(9):1596–1609CrossRefPubMedGoogle Scholar
  82. 82.
    Marcoux J et al (2014) Mass spectrometry defines the C-terminal dimerization domain and enables modeling of the structure of full-length OmpA. Structure 22(5):781–790CrossRefPubMedPubMedCentralGoogle Scholar
  83. 83.
    Betanzos M et al (2002) A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension. Nat Struct Biol 9(9):704–710CrossRefPubMedGoogle Scholar
  84. 84.
    Birkner JP, Poolman B, Kocer A (2012) Hydrophobic gating of mechanosensitive channel of large conductance evidenced by single-subunit resolution. Proc Natl Acad Sci U S A 109(32):12944–12949CrossRefPubMedPubMedCentralGoogle Scholar
  85. 85.
    Bechara C, Robinson CV (2015) Different modes of lipid binding to membrane proteins probed by mass spectrometry. J Am Chem Soc 137(16):5240–5247CrossRefPubMedGoogle Scholar
  86. 86.
    Strop P, Brunger AT (2005) Refractive index-based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci 14(8):2207–2211CrossRefPubMedPubMedCentralGoogle Scholar
  87. 87.
    Salbo R et al (2012) Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers. Rapid Commun Mass Spectrom 26(10):1181–1193CrossRefPubMedGoogle Scholar
  88. 88.
    Jurneczko E et al (2012) Effects of drift gas on collision cross sections of a protein standard in linear drift tube and traveling wave ion mobility mass spectrometry. Anal Chem 84(20):8524–8531CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jeroen F. van Dyck
    • 1
  • Albert Konijnenberg
    • 1
  • Frank Sobott
    • 1
    • 2
    • 3
  1. 1.Biomolecular and Analytical Mass Spectrometry Group, Chemistry DepartmentUniversity of AntwerpAntwerpBelgium
  2. 2.Astbury Centre for Structural Molecular BiologyUniversity of LeedsLeedsUK
  3. 3.School of Molecular and Cellular BiologyUniversity of LeedsLeedsUK

Personalised recommendations