MassIVE MSV000087891

Partial Public PXD027605

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry

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Description

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation plays key roles in altering viral binding/function and infectivity. However, the molecular structures and glycan heterogeneity of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis by conventional bottom-up glycoproteomic approaches. Here, we report the complete structural elucidation of intact O-glycan proteoforms through a hybrid native and denaturing top-down mass spectrometry (MS) approach employing both trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR)-MS. Native top-down TIMS-MS/MS separates the protein conformers of the S-RBD to reveal their gas-phase structural heterogeneity, and top-down FTICR-MS/MS provides in-depth glycoform analysis for unambiguous identification of the glycan structures and their glycosites. A total of eight O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that this hybrid top-down MS approach can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general. [doi:10.25345/C5WJ9H] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: O-Glycoform ; O-glycosylation ; COVID-19 ; SARS-CoV-2 ; Spike protein ; regional binding domain ; top-down mass spectrometry

Contact

Principal Investigators:
(in alphabetical order) 		David S. Roberts, University of Wisconsin-Madison, USA
Ying Ge, University of Wisconsin-Madison, USA
Submitting User: dsroberts

Publications

Roberts DS, Mann M, Melby JA, Larson EJ, Zhu Y, Brasier AR, Jin S, Ge Y.
Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry.
J Am Chem Soc. Epub 2021 Jul 30. DOI: 10.1021/jacs.1c02713.

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