Characterizing Intact Macromolecular Complexes Using Native Mass Spectrometry

  • Elisabetta Boeri Erba
  • Luca Signor
  • Mizar F. Oliva
  • Fabienne Hans
  • Carlo Petosa
Part of the Methods in Molecular Biology book series (MIMB, volume 1764)


Native mass spectrometry (MS) enables the characterization of macromolecular assemblies with high sensitivity. It can reveal the stoichiometry of subunits as well as their two-dimensional interaction network and provide information regarding the dynamic behavior of macromolecular complexes. Here, we describe the workflow to perform native MS experiments. In addition, we illustrate the quality control analysis of proteins using MS in denaturing conditions.

Key words

Macromolecular assemblies Native mass spectrometry Stoichiometry Two-dimensional map of interactions 



We thank Paul Sauer, Jennifer Timm and Daniel Panne for providing the yeast CAF1 complex. We thank the members of the Viral Infection and Cancer Group at the IBS for the helpful discussion. This work used the mass spectrometry platform of the Grenoble Instruct Centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). It was financially supported by the French Infrastructure for Integrated Structural Biology Initiative and by the French National Centre for Scientific Research (CNRS).


  1. 1.
    de Hoffmann E, Stroobant V (2007) Mass spectrometry: principles and applications, 3rd edn. Wiley, New York, NY, p 502Google Scholar
  2. 2.
    Lossl P, van de Waterbeemd M, Heck AJ (2016) The diverse and expanding role of mass spectrometry in structural and molecular biology. EMBO J 35:2634–2657CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kebarle P, Verkerk UH (2009) Electrospray: from ions in solution to ions in the gas phase, what we know now. Mass Spectrom Rev 28:898–917CrossRefPubMedGoogle Scholar
  4. 4.
    Konermann L, Ahadi E, Rodriguez AD, Vahidi S (2013) Unraveling the mechanism of electrospray ionization. Anal Chem 85:2–9CrossRefPubMedGoogle Scholar
  5. 5.
    Cotter RJ (1999) Peer reviewed: the new time-of-flight mass spectrometry. Anal Chem 71:445A–451ACrossRefPubMedGoogle Scholar
  6. 6.
    Makarov A (2000) Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis. Anal Chem 72:1156–1162CrossRefPubMedGoogle Scholar
  7. 7.
    Perry RH, Cooks RG, Noll RJ (2008) Orbitrap mass spectrometry: instrumentation, ion motion and applications. Mass Spectrom Rev 27:661–699CrossRefPubMedGoogle Scholar
  8. 8.
    Sharon M (2013) Biochemistry. Structural MS pulls its weight. Science 340:1059–1060CrossRefPubMedGoogle Scholar
  9. 9.
    Leney AC, Heck AJ (2017) Native mass spectrometry: what is in the name? J Am Soc Mass Spectrom 28:5–13CrossRefPubMedGoogle Scholar
  10. 10.
    Marx V (2016) Proteomics: taking on protein complexes. Nat Methods 13:721–727CrossRefPubMedGoogle Scholar
  11. 11.
    Boeri Erba E, Petosa C (2015) The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes. Protein Sci 24:1176–1192CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Boeri Erba E, Barylyuk K, Yang Y, Zenobi R (2011) Quantifying protein-protein interactions within noncovalent complexes using electrospray ionization mass spectrometry. Anal Chem 83:9251–9259CrossRefPubMedGoogle Scholar
  13. 13.
    Yee AW, Moulin M, Breteau N et al (2016) Impact of deuteration on the assembly kinetics of transthyretin monitored by native mass spectrometry and implications for amyloidoses. Angew Chem Int Ed Engl 55:9292–9296CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    van den Heuvel RH, van Duijn E, Mazon H et al (2006) Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. Anal Chem 78:7473–7483CrossRefPubMedGoogle Scholar
  15. 15.
    Hernandez H, Robinson CV (2007) Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat Protoc 2:715–726CrossRefPubMedGoogle Scholar
  16. 16.
    Kirshenbaum N, Michaelevski I, Sharon M (2010) Analyzing large protein complexes by structural mass spectrometry. J Vis ExpGoogle Scholar
  17. 17.
    Wilm M, Mann M (1996) Analytical properties of the nanoelectrospray ion source. Anal Chem 68:1–8CrossRefPubMedGoogle Scholar
  18. 18.
    Fenn JB, Mann M, Meng CK et al (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246:64–71CrossRefPubMedGoogle Scholar
  19. 19.
    Benesch JL, Ruotolo BT, Simmons DA, Robinson CV (2007) Protein complexes in the gas phase: technology for structural genomics and proteomics. Chem Rev 107:3544–3567CrossRefPubMedGoogle Scholar
  20. 20.
    Snijder J, Rose RJ, Veesler D et al (2013) Studying 18 MDa virus assemblies with native mass spectrometry. Angew Chem Int Ed Engl 52:4020–4023CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Rose RJ, Damoc E, Denisov E et al (2012) High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat Methods 9:1084–1086CrossRefPubMedGoogle Scholar
  22. 22.
    van de Waterbeemd M, Snijder J, Tsvetkova IB et al (2016) Examining the heterogeneous genome content of multipartite viruses BMV and CCMV by native mass spectrometry. J Am Soc Mass Spectrom 27:1000–1009CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Yang Y, Wang G, Song T et al (2017) Resolving the micro-heterogeneity and structural integrity of monoclonal antibodies by hybrid mass spectrometric approaches. MAbs 9:1–8CrossRefGoogle Scholar
  24. 24.
    Kondrat FD, Struwe WB, Benesch JL (2015) Native mass spectrometry: towards high-throughput structural proteomics. Methods Mol Biol 1261:349–371CrossRefPubMedGoogle Scholar
  25. 25.
    Quintyn RS, Zhou M, Yan J, Wysocki VH (2015) Surface-induced dissociation mass spectra as a tool for distinguishing different structural forms of gas-phase multimeric protein complexes. Anal Chem 87:11879–11886CrossRefPubMedGoogle Scholar
  26. 26.
    Harvey SR, Liu Y, Liu W et al (2017) Surface induced dissociation as a tool to study membrane protein complexes. Chem Commun (Camb) 53:3106–3109CrossRefGoogle Scholar
  27. 27.
    Rozen S, Tieri A, Ridner G et al (2013) Exposing the subunit diversity within protein complexes: a mass spectrometry approach. Methods 59:270–277CrossRefPubMedGoogle Scholar
  28. 28.
    Signor L, Boeri Erba E (2013) Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric analysis of intact proteins larger than 100 kDa. J Vis ExpGoogle Scholar
  29. 29.
    Laganowsky A, Reading E, Hopper JT, Robinson CV (2013) Mass spectrometry of intact membrane protein complexes. Nat Protoc 8:639–651CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Hopper JT, Yu YT, Li D et al (2013) Detergent-free mass spectrometry of membrane protein complexes. Nat Methods 10:1206–1208CrossRefPubMedGoogle Scholar
  31. 31.
    Leney AC, McMorran LM, Radford SE, Ashcroft AE (2012) Amphipathic polymers enable the study of functional membrane proteins in the gas phase. Anal Chem 84:9841–9847CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Sobott F, Hernandez H, McCammon MG et al (2002) A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal Chem 74:1402–1407CrossRefPubMedGoogle Scholar
  33. 33.
    Snijder J, Heck AJ (2014) Analytical approaches for size and mass analysis of large protein assemblies. Annu Rev Anal Chem (Palo Alto, Calif) 7:43–64CrossRefGoogle Scholar
  34. 34.
    Morgner N, Robinson CV (2012) Massign: an assignment strategy for maximizing information from the mass spectra of heterogeneous protein assemblies. Anal Chem 84:2939–2948CrossRefPubMedGoogle Scholar
  35. 35.
    Aebersold R, Mann M (2016) Mass-spectrometric exploration of proteome structure and function. Nature 537:347–355CrossRefPubMedGoogle Scholar
  36. 36.
    Loo JA (2000) Electrospray ionization mass spectrometry: a technology for studying noncovalent macromolecular complexes. Int J Mass Spectrom Ion Process 200:175–186CrossRefGoogle Scholar
  37. 37.
    Heck AJ, Van Den Heuvel RH (2004) Investigation of intact protein complexes by mass spectrometry. Mass Spectrom Rev 23:368–389CrossRefPubMedGoogle Scholar
  38. 38.
    Pagel K, Hyung SJ, Ruotolo BT, Robinson CV (2010) Alternate dissociation pathways identified in charge-reduced protein complex ions. Anal Chem 82:5363–5372CrossRefPubMedGoogle Scholar
  39. 39.
    Dyachenko A, Gruber R, Shimon L et al (2013) Allosteric mechanisms can be distinguished using structural mass spectrometry. Proc Natl Acad Sci U S A 110:7235–7239CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    King R, Bonfiglio R, Fernandez-Metzler C et al (2000) Mechanistic investigation of ionization suppression in electrospray ionization. J Am Soc Mass Spectrom 11:942–950CrossRefPubMedGoogle Scholar
  41. 41.
    Annesley TM (2003) Ion suppression in mass spectrometry. Clin Chem 49:1041–1044CrossRefPubMedGoogle Scholar
  42. 42.
    Kastner B, Fischer N, Golas MM et al (2008) GraFix: sample preparation for single-particle electron cryomicroscopy. Nat Methods 5:53–55CrossRefPubMedGoogle Scholar
  43. 43.
    Boeri Erba E, Klein PA, Signor L (2015) Combining a NHS ester and glutaraldehyde improves crosslinking prior to MALDI MS analysis of intact protein complexes. J Mass Spectrom 50:1114–1119CrossRefPubMedGoogle Scholar
  44. 44.
    Caillat C, Macheboeuf P, Wu Y et al (2015) Asymmetric ring structure of Vps4 required for ESCRT-III disassembly. Nat Commun 6:8781CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Barrera NP, Isaacson SC, Zhou M et al (2009) Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nat Methods 6:585–587CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Barrera NP, Robinson CV (2011) Advances in the mass spectrometry of membrane proteins: from individual proteins to intact complexes. Annu Rev Biochem 80:247–271CrossRefPubMedGoogle Scholar
  47. 47.
    Ruotolo BT, Robinson CV (2006) Aspects of native proteins are retained in vacuum. Curr Opin Chem Biol 10:402–408CrossRefPubMedGoogle Scholar
  48. 48.
    Painter AJ, Jaya N, Basha E et al (2008) Real-time monitoring of protein complexes reveals their quaternary organization and dynamics. Chem Biol 15:246–253CrossRefPubMedGoogle Scholar
  49. 49.
    Kelly MA, Vestling MM, Fenselau C, Smith PB (1992) Electrospray analysis of proteins: a comparison of positive-ion and negative-ion mass spectra at high and low pH. Org Mass Spectrom 27:1143–1147CrossRefGoogle Scholar
  50. 50.
    Madler S, Barylyuk K, Boeri Erba E et al (2012) Compelling advantages of negative ion mode detection in high-mass MALDI-MS for homomeric protein complexes. J Am Soc Mass Spectrom 23:213–224CrossRefPubMedGoogle Scholar
  51. 51.
    Allen SJ, Schwartz AM, Bush MF (2013) Effects of polarity on the structures and charge states of native-like proteins and protein complexes in the gas phase. Anal Chem 85:12055–12061CrossRefPubMedGoogle Scholar
  52. 52.
    Chernushevich IV, Thomson BA (2004) Collisional cooling of large ions in electrospray mass spectrometry. Anal Chem 76:1754–1760CrossRefPubMedGoogle Scholar
  53. 53.
    Levy ED, Boeri Erba E, Robinson CV, Teichmann SA (2008) Assembly reflects evolution of protein complexes. Nature 453:1262–1265CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Ahnert SE, Marsh JA, Hernandez H et al (2015) Principles of assembly reveal a periodic table of protein complexes. Science 350:aaa2245CrossRefPubMedGoogle Scholar
  55. 55.
    McKay AR, Ruotolo BT, Ilag LL, Robinson CV (2006) Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. J Am Chem Soc 128:11433–11442CrossRefPubMedGoogle Scholar
  56. 56.
    Keetch CA, Bromley EH, McCammon MG et al (2005) L55P transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers. J Biol Chem 280:41667–41674CrossRefPubMedGoogle Scholar
  57. 57.
    Chevreux G, Atmanene C, Lopez P et al (2011) Monitoring the dynamics of monomer exchange using electrospray mass spectrometry: the case of the dimeric glucosamine-6-phosphate synthase. J Am Soc Mass Spectrom 22:431–439CrossRefPubMedGoogle Scholar
  58. 58.
    Boeri Erba E, Ruotolo BT, Barsky D, Robinson CV (2010) Ion mobility-mass spectrometry reveals the influence of subunit packing and charge on the dissociation of multiprotein complexes. Anal Chem 82:9702–9710CrossRefPubMedGoogle Scholar
  59. 59.
    Sauer PV, Timm J, Liu D et al (2017) Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1. elife 6:e23474CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Elisabetta Boeri Erba
    • 1
  • Luca Signor
    • 1
  • Mizar F. Oliva
    • 1
  • Fabienne Hans
    • 1
  • Carlo Petosa
    • 1
  1. 1.Institut de Biologie Structurale (IBS)Université de Grenoble Alpes, CEA, CNRSGrenobleFrance

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