HDX-MS Analysis of SARS-CoV-2 Spike Ectodomain
This study used hydrogen/deuterium exchange mass spectrometry (HDX-MS) to analyze the conformational dynamics of the SARS-CoV-2 spike ectodomain, demonstrating that including deuterated glycopeptides from its 22 N-glycosylation sites increased sequence coverage from 76% to 84%, thereby enhancing the localization of structural rearrangements and overcoming previous coverage gaps caused by glycosylation.
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) provides precise analysis of a protein’s conformational dynamics across different states, such as heat-denatured versus native protein structures. This technique helps localize regions specifically affected by such conditional changes. Maximizing protein sequence coverage is crucial for high confidence in identifying regions of interest using HDX-MS. However, achieving complete sequence coverage can be challenging due to N-glycosylation sites. In previous HDX-MS analyses, the deuteration of peptides modified by asparagine-bound glycans (glycopeptides) has often not been identified, leading to significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics across many regions of a glycoprotein.
This study reports HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric pre-fusion form, which contains 22 predicted N-glycosylation sites per monomer, both with and without heat treatment. Glycopeptides were identified, and their average isotopic mass shifts from deuteration were calculated. Including the deuterated glycopeptides increased sequence coverage of the spike ectodomain from 76% to 84%. This demonstrated that glycopeptides had been deuterated and improved confidence in results localizing structural rearrangements.
Learnings:
- Analysis and visualization of the conformational dynamics of the SARS-CoV-2 spike glycoprotein
- Explanation of the experimental workflow of hydrogen/deuterium exchange mass spectrometry
- Inclusion of glycopeptides in the analysis of spike conformational dynamics improves visualization detail and fills coverage gaps reported by previous publications
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