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Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry

Nature Protocols volume 2, pages 715–726 (2007)Cite this article

Abstract

The growing number of applications to determine the stoichiometry, interactions and even subunit architecture of protein complexes from mass spectra suggests that some general guidelines can now be proposed. In this protocol, we describe the necessary steps required to maintain interactions between subunits in the gas phase. We begin with the preparation of suitable solutions for electrospray (ES) and then consider the transmission of complexes through the various stages of the mass spectrometer until their detection. Subsequent steps are also described, including the dissociation of these complexes into multiple subcomplexes for generation of interaction networks. Throughout we highlight the critical experimental factors that determine success. Overall, we develop a generic protocol that can be carried out using commercially available ES mass spectrometers without extensive modification.

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Figure 1: Effect of ammonium acetate concentration and added buffer salts on the spectra of alcohol dehydrogenase (ADH).
Figure 2
Figure 3: Effect of pressure on the monomer:tetramer ratio of alcohol dehydrogenase (ADH).
Figure 4: Effect of concentration on the relative intensity of nonspecific oligomers of the tetramer of pyruvate kinase (a) 30 μM (b) 10 μM (c) 1 μM (d) 200 nM.
Figure 5: Mass spectrometry (MS) and MS/MS spectra of a protein–RNA complex, human U1snRNP assembled in vitro (comprising seven Sm core proteins: D1, D2, D3, B, E, F and G, two further proteins U170k and U1A, and synthetic U1snRNA).

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References

  1. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    Article  CAS  Google Scholar 

  2. Phizicky, E.M. & Fields, S. Protein-protein interactions: methods for detection and analysis. Am. Soc. Microbiol. 59, 94–123 (1995).

    CAS  Google Scholar 

  3. Wilm, M.S. & Mann, M. Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last? Int. J. Mass Spectrom. Ion Process 136, 167 (1994).

    Article  CAS  Google Scholar 

  4. Sobott, F., Hernandez, H., McCammon, M.G., Tito, M.A. & Robinson, C.V. A Tandem Mass Spectrometer for Improved Transmission and Analysis of Large Macromolecular Assemblies. Anal. Chem. 74, 1402–1407 (2002).

    Article  CAS  Google Scholar 

  5. Hernandez, H., Dziembowski, A., Taverner, T., Seraphin, B. & Robinson, C.V. Subunit architecture of multimeric complexes isolated directly from cells. EMBO Rep. 7, 605–610 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Synowsky, S.A., van den Heuvel, R.H., Mohammed, S., Pijnappel, P.W. & Heck, A.J. Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex. Mol. Cell. Proteomics 5, 1581–1592 (2006).

    Article  CAS  Google Scholar 

  7. Sharon, M., Taverner, T., Ambroggio, X.I., Deshaies, R.J. & Robinson, C.V. Structural organization of the 19S proteasome lid: insights from MS of intact complexes. PloS Biol. 4, 1314–1323 (2006).

    Article  CAS  Google Scholar 

  8. Ilag, L.L. et al. Heptameric (L12)(6)/L10 rather than canonical pentameric complexes are found by tandem MS of intact ribosomes from thermophilic bacteria. Proc. Natl. Acad. Sci. USA 102, 8192–8197 (2005).

    Article  CAS  Google Scholar 

  9. Elkins, J.M. et al. Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis. Biochem. J. 366, 423–434 (2002).

    Article  CAS  Google Scholar 

  10. Sobott, F., Benesch, J.L., Vierling, E. & Robinson, C.V. Subunit exchange of multimeric protein complexes. Real-time monitoring of subunit exchange between small heat shock proteins by using electrospray mass spectrometry. J. Biol. Chem. 277, 38921–38929 (2002).

    Article  CAS  Google Scholar 

  11. Keetch, C.A. et al. L55P transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers. J. Biol. Chem. 280, 41667–41674 (2005).

    Article  CAS  Google Scholar 

  12. Remaut, H. et al. Donor-strand exchange in chaperone-assisted pilus assembly proceeds through a concerted beta strand displacement mechanism. Mol. Cell. 22, 831–842 (2006).

    Article  CAS  Google Scholar 

  13. Robinson, C.V. et al. Probing the nature of non-covalent interactions by mass spectrometry. A study of protein-CoA ligand binding and assembly. J. Am. Chem. Soc. 118, 8646–8653 (1996).

    Article  CAS  Google Scholar 

  14. Loo, J.A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom. Rev. 16, 1–23 (1997).

    Article  CAS  Google Scholar 

  15. Fernandez de la Mora, J. Electrospray ionization of large multiply charged species proceeds via Dole's charged residue mechanism. Anal. Chim. Acta 406, 93–104 (2000).

    Article  CAS  Google Scholar 

  16. Chernushevich, I.V. & Thomson, B.A. Collisional cooling of large ions in electrospray mass spectrometry. Anal. Chem. 76, 1754–1760 (2004).

    Article  CAS  Google Scholar 

  17. Rostom, A.A. & Robinson, C.V. Detection of the intact GroEL chaperonin assembly by mass spectrometry. J. Am. Chem. Soc. 121, 4718–4719 (1999).

    Article  CAS  Google Scholar 

  18. Gavin, A.C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002).

    Article  CAS  Google Scholar 

  19. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    Article  CAS  Google Scholar 

  20. Gavin, A.C. et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636 (2006).

    Article  CAS  Google Scholar 

  21. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    Article  CAS  Google Scholar 

  22. Verkerk, U.H. & Kebarle, P. Ion-ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues. J. Am. Soc. Mass Spectrom. 16, 1325–1341 (2005).

    Article  CAS  Google Scholar 

  23. Iavarone, A.T., Udekwu, O.A. & Williams, E.R. Buffer loading for counteracting metal salt-induced signal suppression in electrospray ionization. Anal. Chem. 76, 3944–3950 (2004).

    Article  CAS  Google Scholar 

  24. Kapur, A., Beck, J.L., Brown, S.E., Dixon, N.E. & Sheil, M.M. Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA. Protein Sci. 11, 147–157 (2002).

    Article  CAS  Google Scholar 

  25. Schmidt, A., Bahr, U. & Karas, M. Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS. Anal. Chem. 73, 6040–6046 (2001).

    Article  CAS  Google Scholar 

  26. Douglas, D.J. & French, J.B. Collisional focusing effects in radiofrequency quadrupoles. J. Am. Soc. Mass Spectrom. 3, 398–408 (1992).

    Article  CAS  Google Scholar 

  27. Light-Wahl, K.J., Schwartz, B.L. & Smith, R.D. Observation of the noncovalent quaternary associations of proteins by electrospray ionization mass spectrometry. J. Am. Chem. Soc. 116, 5271–5278(1994).

  28. Sobott, F. & Robinson, C.V. Characterising electrosprayed biomolecules using tandem-MS—the noncovalent GroEL chaperonin assembly. Int. J. Mass. Spectrom. 236, 25–32 (2004).

    Article  CAS  Google Scholar 

  29. El-Faramawy, A., Siu, K.W. & Thomson, B.A. Efficiency of nano-electrospray ionization. J. Am. Soc. Mass Spectrom. 16, 1702–1707 (2005).

    Article  CAS  Google Scholar 

  30. Van Berkel, G.J., Zhou, F. & Aronson, J.T. Changes in bulk solution pH caused by the inherent controlled-current electrolytic process of an electrospray ion source. Int. J. Mass Spectrom. Ion Process 162, 55–68 (1997).

    Article  CAS  Google Scholar 

  31. Chernushevich, I.V., Bahr, U. & Karas, M. Nanospray 'taxation' and how to avoid it. Rap. Comm. Mass Spectrom. 18, 2479–2485 (2004).

    Article  CAS  Google Scholar 

  32. Kershaw, N.J. et al. ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activity. Eur. J. Biochem. 269, 2052–2059 (2002).

    Article  CAS  Google Scholar 

  33. Gupta, R., Hamdan, S.M., Dixon, N.E., Sheil, M.M. & Beck, J.L. Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III. Protein Sci. 13, 2878–2887 (2004).

    Article  CAS  Google Scholar 

  34. Benesch, J.L., Aquilina, J.A., Ruotolo, B.T., Sobott, F. & Robinson, C.V. Tandem mass spectrometry reveals the quaternary organization of macromolecular assemblies. Chem. Biol. 13, 597–605 (2006).

    Article  CAS  Google Scholar 

  35. Jurchen, J.C. & Williams, E.R. Origin of asymmetric charge partitioning in the dissociation of gas-phase protein homodimers. J. Am. Chem. Soc. 125, 2817–2826 (2003).

    Article  CAS  Google Scholar 

  36. Tito, M.A., Tars, K., Valegard, K., Hajdu, J. & Robinson, C.V. Electrospray time-of-flight mass spectrometry of the intact MS2 virus capsid. J. Am. Chem. Soc. 122, 3550–3551 (2000).

    Article  CAS  Google Scholar 

  37. McKay, A.R., Ruotolo, B.T., Ilag, L.L. & Robinson, C.V. Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. J. Am. Chem. Soc. 128, 11433–11442 (2006).

    Article  CAS  Google Scholar 

  38. van Breukelen, B., Barendregt, A., Heck, A.J. & van den Heuvel, R.H. Resolving stoichiometries and oligomeric states of glutamate synthase protein complexes with curve fitting and simulation of electrospray mass spectra. Rap. Comm. Mass Spectrom. 20, 2490–2496 (2006).

    Article  CAS  Google Scholar 

  39. Kambach, C. et al. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 96, 375–387 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Matak-Vinkovic, A. Sandercock, M. Sharon and J. Benesch for critical reading of the manuscript and L. Lane for evaluating 'standard' protein complexes. The human U1snRNP sample was kindly donated by D. Pomeranz-Krummel in the group of K. Nagai, MRC Laboratory of Molecular Biology, Cambridge, UK. We thank the Biotechnology and Biological Sciences Research Council (BBSRC) for funding.

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  1. Department of Chemistry, Cambridge University, Lensfield Road, Cambridge, CB2 1EW, UK

    Helena Hernández & Carol V Robinson

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Hernández, H., Robinson, C. Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat Protoc 2, 715–726 (2007). https://doi.org/10.1038/nprot.2007.73

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