tetano
Editor, Senior Moderator
J Am Chem Soc
. 2021 Jul 30.
doi: 10.1021/jacs.1c02713. Online ahead of print.
Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry
David S Roberts[SUP] 1 [/SUP], Morgan Mann[SUP] 2 [/SUP], Jake A Melby[SUP] 1 [/SUP], Eli J Larson[SUP] 1 [/SUP], Yanlong Zhu[SUP] 3 4 [/SUP], Allan R Brasier[SUP] 2 5 [/SUP], Song Jin[SUP] 1 [/SUP], Ying Ge[SUP] 1 2 3 [/SUP]
Affiliations
Abstract
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 the 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.
. 2021 Jul 30.
doi: 10.1021/jacs.1c02713. Online ahead of print.
Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry
David S Roberts[SUP] 1 [/SUP], Morgan Mann[SUP] 2 [/SUP], Jake A Melby[SUP] 1 [/SUP], Eli J Larson[SUP] 1 [/SUP], Yanlong Zhu[SUP] 3 4 [/SUP], Allan R Brasier[SUP] 2 5 [/SUP], Song Jin[SUP] 1 [/SUP], Ying Ge[SUP] 1 2 3 [/SUP]
Affiliations
- PMID: 34328324
- DOI: 10.1021/jacs.1c02713
Abstract
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 the 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.