Electron-Activated Dissociation and Collision-Induced Dissociation Glycopeptide Fragmentation for Improved Glycoproteomics.

Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterization of site-specific glycosylation of proteins remains analytically challenging. Collision-induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but the loss of labile modifications renders CID inappropriate for detailed characterization of site-specific glycosylation. Electron-based dissociation methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localization, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron-activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O- and N-glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localization. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localization confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterization.

Modulation of endoplasmic reticulum stress response pathways by respiratory viruses.

Acute respiratory infections (ARIs) are amongst the leading causes of death and disability, and the greatest burden of disease impacts children, pregnant women, and the elderly. Respiratory viruses account for the majority of ARIs. The unfolded protein response (UPR) is a host homeostatic defence mechanism primarily activated in response to aberrant endoplasmic reticulum (ER) resident protein accumulation in cell stresses including viral infection. The UPR has been implicated in the pathogenesis of several respiratory diseases, as the respiratory system is particularly vulnerable to chronic and acute activation of the ER stress response pathway. Many respiratory viruses therefore employ strategies to modulate the UPR during infection, with varying effects on the host and the pathogens. Here, we review the specific means by which respiratory viruses affect the host UPR, particularly in association with the high production of viral glycoproteins, and the impact of UPR activation and subversion on viral replication and disease pathogenesis. We further review the activation of UPR in common co-morbidities of ARIs and discuss the therapeutic potential of modulating the UPR in virally induced respiratory diseases.

Structure and antigenicity of divergent Henipavirus fusion glycoproteins.

In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiang virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.