Site-specific glycosylation of SARS-CoV-2: Big challenges in mass spectrometry analysis

Abstract

Glycosylation of viral proteins is required for the progeny formation and infectivity of virtually all viruses. It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system. Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS-CoV-2 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site-specific glycosylation of SARS-CoV-2 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.

Keywords: N-glycosylation; O-glycosylation; SARS-CoV-2 glycoprotein; glycoproteomics.

FIGURE 1

Anatomy of the SARS‐CoV‐2 particle showing its structural proteins (adapted from Ganji et al., [53]) with emphasis on the SARS‐CoV‐2 Spike (S) protein with its illustrative protein sequence components: S1 and S2 subunits, and Receptor binding domain (RBD). Also, the most commonly observed N‐ and O‐ glycosylation sites on S protein are depicted

FIGURE 2

Schematic depiction of site‐specific bottom up glycoproteomics workflow used to characterize SARS‐CoV‐2 S protein glycosylation. Different approaches to the workflow and other protocols used are described further in Section 2.2

FIGURE 3

Comparison of HCD tandem mass spectra of the PD‐L1 glycopeptide LFNVTSTLR occupied by a biantennary galili recorded at four different collision energies (NCE: 20, 30, 40, 50) (Sanda et al., [83])

FIGURE 4

Summary of site‐specific N‐glycosylation analysis of SARS‐Cov‐2. The N‐glycan sites and glycan composition at each site are compared among different publications. The compositional analysis of the glycans is shown for each site displaying only the most abundant of each of the three types of N‐glycans: unoccupied sites (gray), high‐mannose (green), hybrid (orange), complex (blue), and paucimannose (yellow). When compositional analysis shows equal abundancy of 2 types of N‐glycans, the two correspondent colors are displayed at the same site. Different sources of the S protein, expression systems, and proteases used for sample digestion are also shown for comparison

FIGURE 5

An example of separation of O‐glycostructures using of ion mobility technique. (Sanda et al., [68])

FIGURE 6

Comparison of software used for N‐glycoproteomics data processing from Hackett et al., [89]. (A) Comparison of glycoproteomics software, and (B) data analysis from Zhang et. al., [105]