In-depth characterization of protein N-glycosylation for a COVID-19 variant-design vaccine spike protein.

COVID-19 is caused by SARS-CoV-2 infection and remains one of the biggest pandemics around the world since 2019. Vaccination has proved to be an effective way of preventing SARS-CoV-2 infection and alleviating the hospitalization burden. Among different forms of COVID-19 vaccine design, the spike protein of SARS-CoV-2 virus is widely used as a candidate vaccine antigen. As a surface protein on the virus envelop, the spike was reported to be heavily N-glycosylated and glycosylation had a great impact on its immunogenicity and efficacy. Besides, N-glycosylation might vary greatly on different expression systems and sequence variant designs. Therefore, comprehensive analysis of spike N-glycosylation is of great significance for better vaccine understanding and quality control. In this study, full characterization of N-glycosylation was performed for a Chinese Hamster Ovary (CHO) cell expressed variant-designed spike protein. The spike protein featured the latest six-proline substitution design together with the incorporation of a combination of mutation sites. Trypsin and Glu-C digestion coupled with PNGase F strategies were adopted, and effective LC-MS/MS methods were applied to analyze samples. As a result, a total of 19 N-glycosites were identified in the recombinant pike protein at intact N-glycopeptide level. Quantitative analysis of released glycan by LC-MS/MS was also performed, and 31 high-abundance N-glycans were identified. Sequencing analysis of glycan was further provided to assist glycan structure confirmation. Moreover, all of the analyses were performed on three consecutive manufactured batches and the glycosylation results on both glycosite and glycans showed good batch-to-batch consistency. Thus, the reported analytical strategy and N-glycosylation information may well facilitate studies on SARS-CoV-2 spike protein analysis and quality studies.

Metal phosphonates based on (4-carboxypiperidyl)-N-methylenephosphonate: in situ ligand cleavage and metamagnetism in Co3(O3PCH2-NHC5H9-COO)2(O3PCH2-NC5H10)(H2O).

Hydrothermal reaction of (4-carboxypiperidyl)-N-methylenephosphonic acid (4-cpmpH3) and zinc sulfate results in compound Zn(O3PCH2-NHC5H9-COO) (). It has a pillared layered structure in which the inorganic layers made up of corner-sharing {CPO3} and {ZnO4} tetrahedra are connected by the organic groups. When cobalt sulfate is allowed to react with 4-cpmpH3 under similar conditions, a proportion of the ligands undergo decarboxylation, leading to the formation of a novel mixed ligated compound Co3(O3PCH2-NHC5H9-COO)2(O3PCH2-NC5H10)(H2O) (). Compound also shows a pillared layered structure with the topology of the inorganic layer similar to that of . The CoII ions in are all tetrahedrally coordinated. Significant difference between the two structures lies in the packing of the layers, e.g....AAA... in while ...AABAAB... in . More interestingly, compound behaves as a metamagnet at low temperature. The critical field is 40.1 kOe at 1.8 K.

Chiral-layered metal phosphonate formed via spontaneous resolution showing dehydration-induced antiferromagnetic to ferromagnetic transformation.

Metal phosphonates M (II){(2-C 5H 4N)CH 2NHCH 2PO 3} (H 2O) [M = Mn ( 1), Cd ( 2)] with chiral-layered structures are obtained by spontaneous resolution using achiral starting materials. The magnetic behavior of 1 is transformed from antiferromagnetic to ferromagnetic upon dehydration.