Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies


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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

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

    CAS  Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

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

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  PubMed  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

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

    Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

Download references


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.

Author information




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.

Corresponding author

Correspondence to Albert J R Heck.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 950 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rose, R., Damoc, E., Denisov, E. et al. High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat Methods 9, 1084–1086 (2012).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing