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

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 is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance 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. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS 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.

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

“…[28][29][30][31][32] Two recent publications have used top-down mass spectrometry to study RBD glycosylation. 33,34 Both of these studies identified O-glycosylation at site T323 of the spike S1 protein and two Nglycosylation sites at N331 and N343, the same sites that were previously identified with bottom-up glycoproteomics. 3,19,20 The study from Roberts et al, 34 used denatured and native top-down analysis to determine the relative abundance of O-glycoforms of the RBD, while the study from Gstöttner et al 33 combined bottom-up, and intact protein analysis with multiple glycosidase enzymatic steps to study the N-and O-linked glycosylation profile of the RBD.…”

“…28,39 Variable amounts of sialic acid groups on the O-and the N-glycan sites have been reported on the RBD. 20,33,34 Separation techniques to resolve these complex RBD glycoforms have not been systematically evaluated to the best of our knowledge. One might expect CZE to separate the RBDs well since there is some variance in charged sialic acid, while HILIC may be able to separate the more neutral N-glycans based on total glycan composition.…”

“…The T323 O-glycan site is decorated with 1-2 sialic acids (the most abundant O-glycan has 2 sialic acids) with Core 1 or Core 2 O-glycan structures. 33,34 The identified Nglycans are complex and have between 0-3 sialic acids. 20,33 This combination of O-and N-glycan compositions leads to the majority of RBDs containing 2-4 sialic acids, 33 a narrower distribution than seen with proteins that separate well with CZE such as AGP (13-19 sialic acids).…”

“…Using the same approach, N-terminal ETD fragments for the Sino Biological WT (Figure S10) explained the extra 85.1 Da by an additional serine residue remained from the signaling peptide sequence, as was previously reported for this RBD. 34 We also identified potential cysteinylation at C538 based on non-reducing intact mass analysis after N-glycan removal (Figure S11) and known information in recent reports. 33 Additionally, we detected an unknown 186 Da mass shift unique to the two Sino Biological RBDs (Figure S11C-D).…”

“…The measured masses were 206 Da heavier for the Sino Biological and 733 Da heavier for the RayBiotech RBDs than expected (after considering the known C-terminal poly-histidine affinity tag), suggesting additional modifications. To better confirm the protein sequence, the N-glycans were removed with PNGase F, the protein was reduced, and then denatured to produce O-glycoforms for direct infusion top-down analysis, similar to the approach used by Roberts et al 34 Removal of N-glycosylation significantly reduced spectral complexity and improved fragmentation for sequencing the protein backbones. After accounting for the expected sequence mass (including purification tags) and the O-glycoforms, the RayBiotech WT and N331Q RBDs were still 613.0 and 610.3 Da heavier than expected, respectively (Figure S7).…”