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High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies

Nature Methods volume 9, pages 1084–1086 (2012)Cite this article

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Abstract

The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution.

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Figure 1: Orbitrap-based mass spectra of intact proteins and protein assemblies.
Figure 2: Orbitrap analyzer allows detection at the sensitivity of individual GroEL ions.
Figure 3: The mass resolution of the Orbitrap analyzer allows small molecules binding to large assemblies to be monitored.

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References

  1. Sharon, M. & Robinson, C.V. Annu. Rev. Biochem. 76, 167–193 (2007).

    Article  CAS  Google Scholar 

  2. Heck, A.J. Nat. Methods 5, 927–933 (2008).

    Article  CAS  Google Scholar 

  3. Shoemaker, G.K. et al. Mol. Cell. Proteomics 9, 1742–1751 (2010).

    Article  CAS  Google Scholar 

  4. Sobott, F., Hernandez, H., McCammon, M.G., Tito, M.A. & Robinson, C.V. Anal. Chem. 74, 1402–1407 (2002).

    Article  CAS  Google Scholar 

  5. van den Heuvel, R.H. et al. Anal. Chem. 78, 7473–7483 (2006).

    Article  CAS  Google Scholar 

  6. Christ, P. et al. Eur. J. Mass Spectrom. (Chichester, Eng.) 10, 469–476 (2004).

    Article  CAS  Google Scholar 

  7. Chernushevich, I.V. & Thomson, B.A. Anal. Chem. 76, 1754–1760 (2004).

    Article  CAS  Google Scholar 

  8. Zhang, Z., Pan, H. & Chen, X. Mass Spectrom. Rev. 28, 147–176 (2009).

    Article  CAS  Google Scholar 

  9. Perry, R.H., Cooks, R.G. & Noll, R.J. Mass Spectrom. Rev. 27, 661–699 (2008).

    Article  CAS  Google Scholar 

  10. Hendrix, R.W. & Johnson, J.E. Adv. Exp. Med. Biol. 726, 351–363 (2012).

    Article  CAS  Google Scholar 

  11. Bochtler, M., Ditzel, L., Groll, M., Hartmann, C. & Huber, R. Annu. Rev. Biophys. Biomol. Struct. 28, 295–317 (1999).

    Article  CAS  Google Scholar 

  12. Krishna, K.A., Rao, G.V. & Rao, K.R. Curr. Protein Pept. Sci. 8, 418–425 (2007).

    Article  CAS  Google Scholar 

  13. Rostom, A.A. et al. Proc. Natl. Acad. Sci. USA 97, 5185–5190 (2000).

    Article  CAS  Google Scholar 

  14. Tito, M.A., Tars, K., Valegard, K., Hadju, J. & Robinson, C.V. J. Am. Chem. Soc. 122, 3550–3551 (2000).

    Article  CAS  Google Scholar 

  15. Makarov, A. & Denisov, E. J. Am. Soc. Mass Spectrom. 20, 1486–1495 (2009).

    Article  CAS  Google Scholar 

  16. Benesch, J.L., Aquilina, J.A., Ruotolo, B.T., Sobott, F. & Robinson, C.V. Chem. Biol. 13, 597–605 (2006).

    Article  CAS  Google Scholar 

  17. Monti, M.C. et al. Nucleic Acids Res. 39, 8052–8064 (2011).

    Article  CAS  Google Scholar 

  18. Rogniaux, H. et al. Anal. Biochem. 291, 48–61 (2001).

    Article  CAS  Google Scholar 

  19. Zhou, M. et al. Science 334, 380–385 (2011).

    Article  CAS  Google Scholar 

  20. Cui, W., Rohrs, H.W. & Gross, M.L. Analyst (Lond.) 136, 3854–3864 (2011).

    Article  CAS  Google Scholar 

  21. Quaite-Randall, E. & Joachimiak, A. Methods Mol. Biol. 140, 29–39 (2000).

    CAS  PubMed  Google Scholar 

  22. Olsen, J.V. et al. Mol. Cell. Proteomics 8, 2759–2769 (2009).

    Article  CAS  Google Scholar 

  23. Michalski, A. et al. Mol. Cell. Proteomics 10, M111.011015 (2011).

    Article  Google Scholar 

  24. Makarov, A. Anal. Chem. 72, 1156–1162 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the PRIME-XS project, Grant Agreement Number 262067, and by the PROSPECTS network (grant HEALTH-F4-2008-201648) both funded by the European Union Seventh Framework Program. The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative, is acknowledged for funding. The authors thank Michael Gröll (Technische Universitat Munchen) for his kind donation of the 20S proteasome sample, Jack Johnson (Scripps Research Institute) for the HK97 capsomer sample, Genmab for the IgG antibody, and Uwe Rickens (Thermo Fisher Scientific) for developing IonDetectionConverter software for the single ion experiments.

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Authors and Affiliations

  1. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Rebecca J Rose & Albert J R Heck

  2. Netherlands Proteomics Center, Utrecht, The Netherlands

    Rebecca J Rose & Albert J R Heck

  3. Thermo Fisher Scientific (Bremen), Bremen, Germany

    Eugen Damoc, Eduard Denisov & Alexander Makarov

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  1. Rebecca J Rose
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  2. Eugen Damoc
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  3. Eduard Denisov
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  4. Alexander Makarov
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  5. Albert J R Heck
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Contributions

R.J.R., E.Da., E.De., A.M. and A.J.R.H. performed the experiments and wrote the manuscript. A.M. supervised the modifications on the Orbitrap mass analyzer. A.M. and A.J.R.H. conceptually designed the work.

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Correspondence to Albert J R Heck.

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Competing interests

E.Da., E.De. and A.M. are employees of Thermo Fisher Scientific, the manufacturer of the Exactive Plus instrument used in this research.

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Rose, R., Damoc, E., Denisov, E. et al. High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat Methods 9, 1084–1086 (2012). https://doi.org/10.1038/nmeth.2208

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