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The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size, and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 methods provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass spectrometry-based qualitative and quantitative N-glycoproteomics.
During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. The viral spike (S) protein mediates both the initial contact with the host cell and the subsequent membrane fusion. Proteolytic cleavage of S at the S2′ site exposes its fusion peptide (FP) as the new N-terminus. By binding to the host membrane, the FP anchors the virus to the host cell. The reorganization of S2 between virus and host then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 μs long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP, we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than 10-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP–membrane binding.