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Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein
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  • Christopher A. Haynes,
  • Theodore R. Keppel,
  • Betlehem Mekonnen,
  • Sarah H. Osman,
  • Yu Zhou,
  • Adrian R. Woolfitt,
  • Jakub Baudys,
  • John R. Barr,
  • D Wang
Christopher A. Haynes
Centers for Disease Control and Prevention
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Theodore R. Keppel
Centers for Disease Control and Prevention
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Betlehem Mekonnen
Centers for Disease Control and Prevention
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Sarah H. Osman
Centers for Disease Control and Prevention
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Yu Zhou
Centers for Disease Control and Prevention
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Adrian R. Woolfitt
Centers for Disease Control and Prevention
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Jakub Baudys
Centers for Disease Control and Prevention
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John R. Barr
Centers for Disease Control and Prevention
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D Wang
Centers for Disease Control and Prevention

Corresponding Author:[email protected]

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Abstract

Rationale. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein’s conformational dynamics across varied states, such as heat-denatured vs. native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. Methods. We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions, if high-energy collision-induced dissociation (HCD) MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans then electron transfer low-energy collision-induced dissociation (EThcD) MS/MS scans of the precursor identified the modified asparagine residue and the glycan’s mass. As in traditional HDX-MS the identified glycopeptides were then analyzed at the MS level in samples labeled with D 2O. Results. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric pre-fusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural re-arrangements. Conclusion. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.
15 Sep 2023Submitted to Rapid Communications in Mass Spectrometry
15 Sep 2023Submission Checks Completed
15 Sep 2023Assigned to Editor
15 Sep 2023Review(s) Completed, Editorial Evaluation Pending
17 Sep 2023Reviewer(s) Assigned
21 Nov 2023Editorial Decision: Revise Minor