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Hydrogen/deuterium exchange mass spectrometry analysis of ribosome-nascent chain complexes to study protein biogenesis at the peptide level

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Abstract

Nascent proteins begin to fold during their synthesis, while still attached to the ribosome. The dynamic nature of ribosome–nascent chain complexes (RNCs) poses a challenge for conventional structural biology approaches, limiting our understanding of dynamic cotranslational events. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a powerful label-free technique for studying the conformational equilibria and refolding of full-length proteins with peptide resolution. However, the large size of the ribosome and the need for stable, highly homogeneous samples have hindered the application of HDX-MS to RNCs. Here we present a strategy for analysing conformational dynamics and interactors of Escherichia coli RNCs using HDX-MS. High-quality RNCs are obtained through the gentle lysis of high-density cultures expressing uniformly stalled ribosomes, followed by ultracentrifugation and tag-based affinity purification. Peptide-resolution information on protein conformational dynamics is obtained by pulse deuterium labeling, quenching with an RNA-compatible low pH buffer and offline digestion with pepsin. Extensive data analysis with use of specific internal controls allows for the confident assignment of mass spectra to specific peptides, ensuring good coverage of the nascent chain and ribosomal proteins. This method provides a valuable complement to existing structural techniques such as cryo-electron microscopy and nuclear magnetic resonance, and enables detailed characterization of large, partially structured nascent chains and their interactions with the ribosomal proteins and molecular chaperones. The protocol takes 1–3 months, from sample preparation and data acquisition to data analysis, and requires standard expertise in cloning and protein purification and intermediate expertise in HDX-MS.

Key points

  • This protocol presents a strategy to isolate Escherichia coli ribosome–nascent chain complexes stabilized via a translation stalling sequence and study their dynamics using hydrogen–deuterium exchange mass spectrometry, enabling detailed characterization of the nascent chains and their interactions with the ribosomal proteins and molecular chaperones.

  • This approach is complementary to other structural approaches such as cryo-electron microscopy and NMR resonance that are not well suited to analyze these large and dynamic assemblies.

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Fig. 1: Schematic summarising the steps involved in analysis of RNCs by HDX-MS.
Fig. 2: Purification and quality control of RNCs.
Fig. 3: Set of purified RNCs sampling β-gal synthesis.
Fig. 4: HDX-MS analysis of β-gal RNC1–646.

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Data availability

The MS data have been described previously24 and deposited to the ProteomeXchange Consortium via the PRIDE partner repository80 with the PXD048642 dataset identifier.

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Acknowledgements

From the Francis Crick Institute, we thank S. Mouilleron for preparing HRV 3C protease, C. Soudy for help with preparing immobilized pepsin, S. Maslen and M. Skehel for maintenance of the MS instrument, S. Shivakumaraswamy for purified full-length β-galactosidase, and all members of the Protein Biogenesis Laboratory for useful discussion. D.B. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2025), the UK Medical Research Council (CC2025), the Wellcome Trust (CC2025) and by UK Research and Innovation (UKRI) under the UK government’s Horizon Europe funding guarantee (FoldingMap, EP/X020428/1). J.R.E. acknowledges funding from the National Institutes of Health (R01-CA233978) and the James L. Waters Chair in Analytical Chemistry.

Author information

Author notes

  1. Grant A. Pellowe

    Present address: Aston Institute for Membrane Excellence, Aston University, Birmingham, UK

  2. These authors contributed equally: Alžběta Roeselová, Aleksandra Pajak, Thomas E. Wales, Grant A. Pellowe.

Authors and Affiliations

  1. Protein Biogenesis Laboratory, The Francis Crick Institute, London, UK

    Alžběta Roeselová, Aleksandra Pajak, Grant A. Pellowe & David Balchin

  2. Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA

    Thomas E. Wales & John R. Engen

  3. Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK

    Svend Kjær

Authors
  1. Alžběta Roeselová
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Contributions

A.R. contributed to the optimization of the RNC purification protocol and HDX-MS protocol. A.P. optimized the RNC purification protocol. T.E.W., G.A.P. and J.R.E. optimized the HDX-MS protocol. A.R., A.P. and T.E.W. performed the supporting experiments and analyzed the data. S.K. optimized the muGFP-clamp resin preparation protocol. A.R. wrote the manuscript with input from all other authors. D.B. conceived and supervised the project.

Corresponding authors

Correspondence to John R. Engen or David Balchin.

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Key references

Wales, T. E. et al. Nat. Struct. Mol. Biol. 31, 1888–1897 (2024): https://doi.org/10.1038/s41594-024-01355-x

Roeselová, A. et al. Mol. Cell 84, 2455–2471.e8 (2024): https://doi.org/10.1016/j.molcel.2024.06.002

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Roeselová, A., Pajak, A., Wales, T.E. et al. Hydrogen/deuterium exchange mass spectrometry analysis of ribosome-nascent chain complexes to study protein biogenesis at the peptide level. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01279-w

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