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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.

Glycosylation is one of the most important types of PTMs that could potentially impact protein structure, orientation, binding affinity, and metabolism [6]. It involves the covalent attachment of different types of glycans to specific sites on protein structures. Viral envelope proteins are often ornamented by glycans that can account for up to half of the molecular weight of these glycoproteins [7]. Despite the numerous types of glycosylation, N-and mucin-type O-linked glycosylation are the most widely exploited in viral research [8]. Prominent examples include the heavily glycosylated gp120 glycoprotein in HIV, the Ebola virus glycoprotein modified by a very high glycan content, and the HIV-1 glycoprotein gp160 that is glycosylated by the addition of multiple N-linked glycans [7,[9][10][11].
The N-linked glycosylation takes place on the nitrogen atom of the asparagine amide side chain, where this asparagine (N) residue is located within the sequence -N-X-T/S-(where X ≠ proline) [12][13][14].
The O-linked glycosylation is usually meant to attach the sugar moiety to the oxygen of the side chains of serine and/or threonine residues within the amino acid sequence [15][16][17]. Both types of site-specific glycosylation have been shown to affect viral glycoprotein secretion and function [8].
As the virus hijacks the host cellular machinery for replication, it subsequently uses the host glycosylation capabilities for the production of viral proteins. Thus, the viral surface antigens may encompass familiar host glycans, which can thereby change the ability of the host to recognize the virus and stimulate the immune response [18][19][20].
In addition, certain viruses are able to induce changes in the expression levels of host glycosyltransferases (enzymes that catalyze the glycosidic bond). For example, herpes viruses can induce the expression of host fucosyltransferases leading to the expression of sLex or Ley antigens [21,22]. Another example is the shift of the total glycoprofile of HCV-infected hepatoma cells toward more fucosylated, sialylated, and a more complex N-glycan structures [23]. Furthermore, the changes in expression levels of host glycosylation enzymes may indicate occurrence of specific structural or functional modifications or highlight the demand for increased glycosylation capacity to support viral protein glycosylation.
In this review, we examined the most recent advancements in structural identification and quantitation of viral glycosylation. Given the ongoing pandemic and the high demand for protein and structure analysis, we mainly focused on an in-depth analysis of SARS-CoV-2. Also, we highlighted the most intricate challenges in the determination of site-specific glycosylation associated with viral spike protein.

VIRAL GLYCOPROTEINS AND THEIR FUNCTIONS
Glycosylation of viral proteins is required for the progeny formation and infectivity of many viruses [31,34]. Furthermore, distinct glycans play pivotal roles in various stages of the viral cycle [34,35].
For example, glycans on viral entry proteins are greatly involved in the modulation of receptor binding and entry [36]. A prominent example is the influenza viruses that attach to glycans on cellular surface glycoproteins. Hemagglutinin (HA) and neuraminidase (NA) are the surface glycoproteins of influenza viruses, which interact with the terminal sialic acid (SA) of the host cell surface glycoproteins. NA can cleave the SA residues of host mucin to gain access to the epithelial cells, playing a secondary role in helping viruses to enter host cells [37]. On the other hand, NA can also cleave SA residues from glycoproteins of the enveloped virus itself and enhance infectivity by preventing aggregation of viral particles [38]. Host-cell dependent glycosylation of HA and NA has also proven to be critical. Glycosylation in the HA stalk region is important for protein folding and trafficking, and for pH stability [39,40], while the extent of glycosylation near the HA receptor-binding site alters its affinity for SA-containing receptors [41,42]. Glycosylation near the cleavage of HA site also modulates virus pathogenicity [43]. While the role of glycosylation of NA is less well understood, N-linked glycosylation is important for functional NA, and lack of NA glycosylation increases neurovirulence of the A/WSN/33 IAV strain in mice [44]. Besides, HA N-glycosylation affects T cell activation and cytokine production and thus promote immune evasion [45]. This and other types of immune evasion have been reported for different glycoproteins in different viruses. In fact, it is strongly believed that the high levels of glycosylation serve primarily as a protective shield against the host's immune system [46,47].
While the innate immune system is constantly evolving a range of strategies to combat glycosylated epitopes of serious pathogens, mutations could lead to failures in the immune reactions. Alterations on viral glycoproteins can significantly impact viral characteristics, including the extent of protein glycosylation, which may jeopardize efficacy of existing vaccines [45,48]. Moreover, antigen glycosylation complicates the development of vaccines and antibody-based therapies.
Glycans represent structural features that are not encoded in the gene sequence and yet play a crucial role in immune recognition affecting vaccine designs. Such vaccine candidates are often expressed in cell lines that do not recapitulate the glycosylation pattern on native pathogens, and potentially do not elicit biologically relevant immune responses. Therefore, it is important to identify common glycosylation patterns for translational applications. This has been particularly important in the study of SARS-CoV-2 spike (S) protein.

SARS-CoV-2 spike protein glycosylation
SARS-CoV-2 initiates the infection cycle by binding to Angiotensin-Converting Enzyme II (ACE2) on the host epithelial cells in the respiratory tract. This leads to viral penetration, domination of the host cell's biological machinery, multiplication, and maturation of the virus [49][50][51][52]. Figure 1 demonstrates SARS-CoV-2 as represented by Ganji F I G U R E 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 et al., [53]. There are four structural proteins encoded by SARS-CoV-2 genome: the spike (S) glycoprotein, the membrane (M) protein, the envelope (E) protein, and the nucleocapsid (N) protein.
Like many viruses, SARS-CoV-2 launches its cellular invasion through its heavily glycosylated S protein [54][55][56][57][58]. Despite the relatively modest contribution of the glycans to the total molecular weight of the S trimer of the spike protein, they shield approximately 40% of the protein surface. The three-dimensional structure of the S protein shows that the protein surface is extensively shielded from antibody recognition by glycans, with the notable exception of the ACE2 receptor binding domain [59]. The S protein encompasses two protein subunits, S1 and S2. While the S1 subunit is the ACE2 receptor binding domain and modulates receptor recognition, S2 subunit orchestrates cellular adhesion and membrane fusion [60]. The viral S protein of SARS-Cov-2 has also been the ultimate target for vaccine production [61]. In fact, spike proteins are often used as immunogens for vaccines to generate neutralizing antibodies and are frequently targeted for inhibition by small molecules that might block host receptor binding and/or membrane fusion.
Glycosylation is a heterogenic process that depends on many factors, including age, underlying disease and ethnicity; therefore, assessing the glycosylation profile may be correlated to the observed differential susceptibilities among individuals to COVID-19 [62][63][64][65].
While overall shielding of the underlying protein surface does not appear to be highly sensitive to glycan microheterogeneity, it could impact the innate immune response by altering the ability of collectins and other lectins of the immune system to effectively bind to the S glycoprotein and neutralize the virus. Also, it may impact the adaptive immune response by altering the number of viable human leucocyte antigen (HLA) [66].
A summary of the identified O-glycan sites is displayed in Table 1

Strategies used for the analysis of viral spike glycoprotein
Various strategies have been recruited to analyze and confirm the nature, the structure and exact location of glycans on viral spike proteins. These strategies involve the sample preparation, the chromatographic separation, mass spectrometry techniques, and bioinformatics analysis.
The sample preparation usually involved the use of glycan releasing enzymes (such as PNGase F for N-glycans) as well as proteases (such as trypsin) with or without enrichment depending on the glycopeptide stoichiometry. Hydrophilic interaction liquid chromatography (HILIC), lectin affinity chromatography, and graphitized carbon chromatography were widely adopted for enrichment of glycopeptides. Figure 2 demonstrates the workflow for the sample preparation and data acquisition.
The separated glycans or glycopeptides are then analyzed by tandem MS/MS to determine glycosite location and glycan structure of

2.2.2
Glycosylation analysis using mass spectrometers with various instruments and different resolving power SARS-CoV-2 S protein glycosylation was predominantly analyzed only by high-resolution mass spectrometers. In most cases, it was an orbitrap based analyzer working with a resolution of 60,000 to 120,000 or a Time Of Flight (TOF) based analyzer which was used in very few cases of the top-down approach [78,84]. The use of high and ultrahigh resolving power allowed scientists to effectively analyze 22 glycosylation sites in the mixture of digested peptides and glycopeptides. Targeted multiple-reaction monitoring (MRM) and parallel reaction monitoring (PRM) methodology were used for detection and quantification of spike protein fragments in biological fluids such as saliva or extracted nasal swabs [85,86]. Although these methodologies were only reported for nonglycosylated peptide analysis, we envision these will be implemented for spike protein glycosylation analysis of SARS-CoV-2 S protein in the near future.

2.2.3
Data processing software The results were consistent despite the minor variation in a few aspects like using different proteolytic enzyme combination, multiple fragmentation settings, different processing softwares, using ion mobility for separation of glycan isomers [68,78] or using ETD fragmentation for occupied glycosite assignment [68,92]. Top-down approach was exclusively used in the analysis of O-glycosylation of RBD domain since it has only two potential glycosites and allows effective TOP-down analysis of O-glycosylation.

N-glycosylation depends on different source of material
The N-glycosylation of SARS-CoV-2 S proteins has been investigated extensively in the literature. Several studies used recombinant S proteins produced in different expression systems [58,[68][69][70][93][94][95]. This is mostly due to the difficulties inherent to the study of the wild type viral proteins. Human embryonic kidney cell line HEK 293 was the most frequently used expression system to produce the recombinant S protein (Figure 4). Some studies compared the recombinant S protein expressed in HEK cells with protein overexpressed in insect cells [93,95]. As expected, N-glycosylation from different expression systems yielded variable results. Figure 4 describes the different expression systems, source of protein, used proteases and references for the various N-glycosylation analysis. The blue color indicates a complex glycan structure, green refers to a high mannose glycan, orange to hybrid glycans, and yellow to paucimannose glycans.
A total of 3 research groups focused on analysis of S1 subunit encompassing the RBD domain which was overexpressed in HEK 293 cell line [93,95,96], while a single study used S1 subunit isolated from virion cultivated on Calu-3 cells [97]. The three studies of S1 subunit reported N603 mostly occupied by high mannose glycan [95].
Interestingly, a total of 11 out of 16 studies demonstrated that N234 was occupied by oligomannose moiety, while the remaining groups concluded that N234 is decorated with a complex glycan structure which has a direct impact on receptor binding [93][94][95][96]. To note that three of the studies reporting complex glycan structures at N234 used only the S1 subunit expressed in HEK 293 cells, instead of the full size (S1+S2 subunits) spike protein construct. In addition, Brun et al., showed that almost one third of the glycosites (including N234) is occupied by oligomannose structures in the S1 subunit, isolated from virion grown on Calu-3 cells. Three research groups used insect-cell lines for the production of SARS-CoV-2 spike glycoprotein [67,93,95]. It is known that insect cells produce predominantly short version of N-gycans also known as paucimannose glycans. These glycan type was identified on almost all N-glycosylation sites on these studies. Wang  Determination of the expression system independent of the glycosylation events can help understand protein sequence and structure.
To the best of our knowledge, there is no analysis of glycosylation of spike glycoprotein isolated from real patient samples.
Detailed knowledge of S protein glycosylation it's not only important for vaccine development, but also to understand its role on receptor binding. It was recently shown, that N-glycans of different types can have different effects on interaction with receptors [84]. In this study, we showed how S1 protein expressed in different expression models (and hence different major N-glycosylation types) including baculovirus-insect, Chinese hamster ovarian (CHO) cells and two variants of HEK 293 cells, imposed different binding affinities to ACE2.

O-Glycosylation on different proteoforms and different used techniques
One of the important aspects of spike glycoproteins analysis was the form (partial sequence or mutation) of glycoprotein for overexpression.
As mentioned above, Wang et al., [93] Tian et al., [96] and Zhang et al., [95] overexpressed subunit S1 containing receptor binding domain (RBD). Several groups described analysis of protein which has mutated furin cleavage sites such as R683A, R685A [68,92]; R683S, R685S [70] and R683G, A684S, and R685G [58]. Spike protein is overexpressed as one intact unit. Many studies described glycosylation analysis of F I G U R E 5 An example of separation of O-glycostructures using of ion mobility technique. (Sanda et al., [68]) protein overexpressed as trimeric complex similar to real viral spike complex [94]. Table 1  have an influence on the binding of spike protein to ACE2 receptor [52,84]. This information is listed in column "RBD" sites in Table 1. Another physiologically important O-glycosylation region was predicted to be near the furin cleavage site. This polybasic furin cleavage sequence is specific for human SARS-CoV-2 spike glycoprotein [52]. There are three predicted O-glycosites near this site: S673, T678, and S686.
A total of eight reports described T678 occupied sites which is only two amino acid from the furin cleavage site. Also, Gao et al., showed occupied S686 in his report. There was no described identification of occupied S673. In addition, Zhang et al., disclosed occupancy of sites T676 and S680 which were not predicted to be occupied [100].
Mutations of T676 and T678 were used in recent study that confirmed influence of O-glycosylation on furin cleavage efficiency [100].
Some publications used EthcD fragmentation to efficiently assign Oglycosites [68,73]. The rest of the publication mostly used beam type

2.2.7
Source of data variability between laboratories, instrumentation, and methodologies It is important to note that the identified glycoforms of the over-  [96,97,99]. The rest of these publication described glycosylation of overexpressed protein in human embryonic kidney cells or insect-cell expression systems. As expected, the glycosylation composition strongly influenced by the expression system as well as the number of passages [90].

F I G U R E 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] In addition, the type of digestion, LC-MS system as well as the software used for identification and quantification could be a source of interlaboratory variation when describing glycosylation of SARS-CoV-2 spike glycoprotein. A recent interlaboratory study was focused on examining the variations of data processing and evaluation. This study disclosed significant differences in observation of the number of glycoforms as well as composition with various software, evaluation criteria, and experience of researchers [102]. Figure 4 epitomizes the N-glycosylation analysis with information about the source of analyzed material, digestion system used, and glycan identification. As mentioned above Hackett et al., compared four different types of software and the results varied significantly ( Figure 6) [89].In their previous work he used Watanabe et al., data [70] and his software tool named GlycReSoft [89]. They observed similar quantities and glycan distribution as their original work but identified many sulfated glycans especially on the site N1074 (6 out of major 30) and penta-antennary or polyLacNAc containing glycopeptides. This indicates that the results strongly depend on the glycan composition of database used for the study. Figure 6 represents the various types of software and their corresponding outcomes. The N-linked glycans from the native spikes glycans are very similar to that of the recombinant glycoprotein glycans [96].
Differences in data are not only influenced by experimental variation, but also by data misinterpretation as well. For example, bioinformatics mass data analysis by Krishnan et al., has mixed nonidentified N-glycosites in some cases as unoccupied [103]. Furthermore, Segreto et al., misinterpred O-glycosylation data obtained by EthcD as data obtained by HCD fragmentation with modulated collision energy [104].

CONCLUDING REMARKS AND OUTLOOK
In this review, we collected and compared most of results on glycosylation of SARS-CoV-2. We showed the discrepancies found on

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.