Academic Journal
Hydrogen/deuterium exchange mass spectrometry analysis of ribosome-nascent chain complexes to study protein biogenesis at the peptide level.
| Τίτλος: | Hydrogen/deuterium exchange mass spectrometry analysis of ribosome-nascent chain complexes to study protein biogenesis at the peptide level. |
|---|---|
| Συγγραφείς: | Roeselová A; Protein Biogenesis Laboratory, The Francis Crick Institute, London, UK., Pajak A; Protein Biogenesis Laboratory, The Francis Crick Institute, London, UK., Wales TE; Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA., Pellowe GA; Protein Biogenesis Laboratory, The Francis Crick Institute, London, UK.; Aston Institute for Membrane Excellence, Aston University, Birmingham, UK., Kjær S; Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK., Engen JR; Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA. j.engen@northeastern.edu., Balchin D; Protein Biogenesis Laboratory, The Francis Crick Institute, London, UK. david.balchin@crick.ac.uk. |
| Πηγή: | Nature protocols [Nat Protoc] 2026 May; Vol. 21 (5), pp. 2271-2300. Date of Electronic Publication: 2026 Jan 12. |
| Τύπος έκδοσης: | Journal Article; Research Support, N.I.H., Extramural; Research Support, N.I.H., Intramural |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Nature Pub. Group Country of Publication: England NLM ID: 101284307 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1750-2799 (Electronic) Linking ISSN: 17502799 NLM ISO Abbreviation: Nat Protoc Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: London, UK : Nature Pub. Group, 2006- |
| Ιατρικοί όροι (MeSH): | Peptides*/chemistry , Peptides*/isolation & purification , Peptides*/metabolism , Ribosomes*/chemistry , Ribosomes*/metabolism , Hydrogen Deuterium Exchange-Mass Spectrometry* , Protein Biosynthesis*, Escherichia coli Proteins/biosynthesis ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/isolation & purification ; Protein Conformation ; Protein Folding ; Ultracentrifugation |
| Περίληψη: | 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. (© 2026. Springer Nature Limited.) |
| Competing Interests: | Competing interests: The authors declare no competing interests. |
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| Grant Information: | FC001985 United Kingdom WT_ Wellcome Trust; R01-CA233978 U.S. Department of Health & Human Services | National Institutes of Health (NIH); CC2025 United Kingdom CRUK_ Cancer Research UK; CC2025 United Kingdom MRC_ Medical Research Council |
| Substance Nomenclature: | 0 (Peptides) 0 (Escherichia coli Proteins) |
| Entry Date(s): | Date Created: 20260112 Date Completed: 20260715 Latest Revision: 20260813 |
| Update Code: | 20260813 |
| PubMed Central ID: | PMC7619191 |
| DOI: | 10.1038/s41596-025-01279-w |
| PMID: | 41526491 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1750-2799 |
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| DOI: | 10.1038/s41596-025-01279-w |