The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor

Abstract

Graphical abstract Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community—the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native MS as a means of characterizing its interactions with both the host cell–surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work, we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.

Molecular architecture and platelet-activating properties of small immune complexes assembled on intact heparin and their possible involvement in heparin-induced thrombocytopenia

“…Once these two dimensions become comparable, the growth of the complex stops, since neither GAG nor protein components of the complex will have any vacant high-charge density segments left to support further growth. This model is consistent with the notion of the ULC composition conforming to 1:1 stoichiometry, and our rough estimations of the ULC mass based on the model that correlates ionic m/z value and charge (z) in native MS 46 yield ca. 3 MDa (Supplementary Material), suggesting that the number of PF4 tetramers in a single ULC particle exceeds 50.…”

“…The unresolved ionic signal in the mass spectrum (m/z range 25,000-45,000) was used to estimate the average ULC mass (3.0±1.5 MDa) following the approach described elsewhere. 1 Briefly, the mass estimate of ULCs is made using the empirical relationship between the average ionic charge ( . ) and the solvent-exposed surface area of the corresponding species in solution ( ):…”

“…Platelet factor 4 (PF4) is a chemokine that is secreted from activated platelets 1 and involved in a variety of physiological processes, ranging from coagulation and tissue repair to innate immune response. 2 It has gained particular notoriety due to its role in triggering a range of (auto)immune hematologic pathologies, the first of which -heparin-induced thrombocytopenia (HIT) -was initially reported sixty-five years ago.…”

Rapid Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches and Multivariate Analysis

“…The similar trend in differences in glycoform distribution was observed by Tamara et al, who compared the glycosylation patterns of desialylated isoforms of Hp 1-1 and Hp 2-2 phenotypes. 40 In order to evaluate the consistency of the results of XP-RC/ MS measurements with the protein mass profiles extracted from the intact MS measurements, the mass distributions of the intact H-L-L-H molecules were calculated for each Hp phenotype by applying a conventional convolution algorithm 41 to the XP-RC/MS data. Figure S6 in the Supporting Information shows the juxtaposition of the mass distributions of the H-L-L-H species in both phenotypes deduced from the XP-RC/MS and native MS data.…”

Proteomics‐based mass spectrometry profiling of SARS‐CoV‐2 infection from human nasopharyngeal samples

“…Two studies from a single group of researchers used recombinant forms of human ACE2 receptor and the S‐protein to perform native MS and molecular modeling. Using a combination of these advanced techniques, they first showed that heparin plays a major disruptor of the SARS‐CoV‐2 interaction with the host cell receptor (Yang, Hughes, et al, 2020 ) and then evaluated the interactions between S1 domain of S‐protein with the ACE2 ectodomain (Yang et al, 2021 ). In addition to this, another group of researchers characterized the functional relevance of SARS‐CoV‐2 N‐protein and main protease using native MS.…”