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High-fidelity mass analysis unveils heterogeneity in intact ribosomal particles

Nature Methods volume 14, pages 283286 (2017) | Download Citation

Abstract

Investigation of the structure, assembly and function of protein–nucleic acid macromolecular machines requires multidimensional molecular and structural biology approaches. We describe modifications to an Orbitrap mass spectrometer, enabling high-resolution native MS analysis of 0.8- to 2.3-MDa prokaryotic 30S, 50S and 70S ribosome particles and the 9-MDa Flock House virus. The instrument's improved mass range and sensitivity readily exposes unexpected binding of the ribosome-associated protein SRA.

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Acknowledgements

The authors would like to thank P. Lössl and F. Liu (Utrecht University) for assistance in the bottom-up LC-MS/MS analysis. M.v.d.W., K.L.F. and A.J.R.H. are funded by the large-scale proteomics facility Proteins@Work (Project 184.032.201) embedded in the Netherlands Proteomics Centre and supported by the Netherlands Organization for Scientific Research (NWO). M.v.d.W. and A.J.R.H. are also supported by a Projectruimte grant (12PR3303-2) from Fundamenteel Onderzoek der Materie (FOM). A.M. and A.J.R.H. acknowledge additional support through the European Union Horizon 2020 program FET-OPEN project MSmed, Project 686547. A.R. is supported by a University of Texas Medical Branch (UTMB) startup fund and the Texas Rising STARs Award from the University of Texas System.

Author information

Author notes

    • Michiel van de Waterbeemd
    •  & Kyle L Fort

    These authors contributed equally to this work.

Affiliations

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

    • Michiel van de Waterbeemd
    • , Kyle L Fort
    • , Alexander Makarov
    •  & Albert J R Heck
  2. Netherlands Proteomics Center, Utrecht, the Netherlands.

    • Michiel van de Waterbeemd
    • , Kyle L Fort
    •  & Albert J R Heck
  3. Thermo Fisher Scientific, Bremen, Germany.

    • Dmitriy Boll
    • , Maria Reinhardt-Szyba
    •  & Alexander Makarov
  4. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.

    • Andrew Routh
  5. Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas, USA.

    • Andrew Routh

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Contributions

M.v.d.W., K.L.F., D.B. and M.R.-S. performed the experiments. M.v.d.W., K.L.F., A.M. and A.J.R.H. wrote the manuscript. A.R. provided FHV material. A.M. and A.J.R.H. supervised the modifications of the Orbitrap mass analyzer. A.M. and A.J.R.H. designed the study.

Competing interests

D.B., M.R.-S. and A.M. are employees of Thermo Fisher Scientific, the manufacturer and supplier of Orbitrap-based mass spectrometers.

Corresponding author

Correspondence to Albert J R Heck.

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    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Tables 1–4

Excel files

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    Supplementary Data

    List of proteins identified in ribosome preparations through bottom-up LC-MS/MS analysis and their intensity-based absolute quantification value.

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DOI

https://doi.org/10.1038/nmeth.4147

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