Dataset Information

Identification, mapping and relative quantitation of SARS-CoV-2 Spike glycopeptides by Mass-Retention Time Fingerprinting.

ABSTRACT: We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using a LC-TOF mass spectrometer, requires no specialized knowledge of glycan analysis and exploits the differential resolving power of reverse phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. Rather than a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike glycopeptides. This database allows any accurate mass LC-MS laboratory to reliably identify and quantify Spike glycopeptides from a single overnight elastase digest in less than 90 minutes.

SUBMITTER: Chalk R

PROVIDER: S-EPMC8333269 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Publications

Identification, mapping and relative quantitation of SARS-CoV-2 Spike glycopeptides by Mass-Retention Time Fingerprinting.

Chalk Rod R Greenland William E P WEP Moreira Tiago T Coker Jesse J Mukhopadhyay Shubhashish M M SMM Williams Eleanor E Manning Charlotte C Bohstedt Tina T McCrorie Rama R Fernandez-Cid Alejandra A Burgess-Brown Nicola A NA

Communications biology 20210803 1

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using a LC-TOF mass spectrometer, requires no specialized knowledge of glycan analysis and exploits the differential resolving power of reverse phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing ...[more]

PMID: 34345007

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Project description:RationaleHydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus 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.MethodsWe 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 MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation 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 D2 O.ResultsWe report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion 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 rearrangements.ConclusionInclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

Dataset Information

Identification, mapping and relative quantitation of SARS-CoV-2 Spike glycopeptides by Mass-Retention Time Fingerprinting.

Publications

Identification, mapping and relative quantitation of SARS-CoV-2 Spike glycopeptides by Mass-Retention Time Fingerprinting.

Similar Datasets

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