July 23, 2024

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Analyse Glycan Shield to accelerate the development of new therapies

Analyse Glycan Shield to accelerate the development of new therapies

Analyse Glycan Shield to accelerate the development of new therapies. Although vaccines are considered to be one of the effective means to prevent and control infectious diseases, the “highly glycosylated phenomenon” of the virus can cause the virus to produce a variety of mutations. These glycosylation sites are like dense “shields” to help the virus perform. Virulence, successfully survived the retrieval of the human immune system, disrupted the battle of vaccines to evoke an immune response to snipe the virus. The existence of the virus “Glycan Shield” structure makes the development of vaccines very difficult. Today, the editor will share with you the combination of tools in Waters’ glycosylation analysis process to help scientists quickly and accurately perform glycosylation analysis and accelerate the development of new therapies.

Case one: Understand the distribution of SARS-CoV-2 spike proteoglycans

Effective therapy development depends on a deep understanding of the SARS-CoV-2 spike protein structure and target function. Waters scientists identified 42 major glycan peaks based on HILIC retention time, glucose unit (GU) value and accurate quality information: 11 non-glycosylated glycans, 29 fucosyl glycans, and the other Two peaks were designated as dilactose. This strange finding needs to be confirmed by MS/MS and exogenous glycosidase array. These glycans can be further divided into 3 categories, including 6 high mannose glycans, 6 hybrid glycans, and 30 complex glycans.

Analyse Glycan Shield to accelerate the development of new therapies

Comprehensive characterization and monitoring of recombinant proteins through peptide maps are often used to identify and track post-translational modifications. Scientists at Waters applied peptide map analysis to analyze recombinant SARS-CoV-2 spikes based on the BioAccord LC-MS system Protein to confirm sequence information and initially assess site-specific glycine heterogeneity.

Characteristic N-sugars are identified with confidence, especially for the abundant oligosaccharide type N234 sugar, which is related to the shielding of receptor binding sites. The O-glycosylation of T323 has also been confirmed by recent reports. These data show that the BioAccord-based peptide map analysis process has the potential to map complex and recombinant glycosylation sites/glycoproteins. Different enzyme treatments can also be tried to maximize coverage.

Streamlined and simplified peptide mapping analysis reveals glycosylation site occupation

Quickly and highly sensitively characterize the N-sugar SARS-COV-2 spike protein on the spike protein. Peptide map after treatment A) Trypsin digestion B) Trypsin + 1hr PNGase F (The illustration shows the UNIFI treatment Sequence coverage)

More than 90% of previously reported glycosylation sites were successfully assigned

Picture of an N-glycopeptide assignment at site N234 based on high-energy fragmentation data

Picture an O-glycopeptide assignment at site T323 based on high energy fragment data

Waters solution for SARS-COV-2 spike protein analysis and characterization

Case two: Analyze the glycan composition and structure spectrum of LASV glycoprotein complex (GPC)

Glycosylation of common virus envelopes, glycosylation sites play an important role in viruses

So far, there is still no licensed vaccine against Lassa virus (LASV). Whether it is during natural infection or through vaccination attempts, it is believed to be due to extensive glycosylation in the LASV glycoprotein complex (GPC) surface. In order to quantitatively evaluate the composition of LASV GPC glycosylation, Dr. Yasunori Watanabe from the Institute of Glycobiology, University of Oxford and others analyzed the fluorescent labeling of N-glycans released by enzymes, using hydrophilic interaction chromatography-ultra-high performance liquid chromatography (HILIC- UPLC) analysis, followed by ion mobility-electrospray ionization mass spectrometry (IM-ESI MS) SYNAPT system to determine the precise structure of each glycan. Consistent with the data obtained by UPLC, IM-ESI MS identified a major mannose type population.

Although LASV GPC constitutes a key therapeutic target, vaccine design efforts have been hampered by host-derived glycosylation. Yasunori Watanabe et al. conducted a comprehensive composition study on LASV glycosylation. The existence of a large number of oligosaccharide structures on LASV GPC is of great significance to the development and production of recombinant vaccine candidates. This study also revealed that the diversification of amino acids mainly occurs in the sugar-free area of ​​LASV GPC, which provides important clues to which sites may be most susceptible to antibody immune responses. Similar to template-based vaccinology methods for “escape” pathogens (such as HIV-1), these regions may be viable targets for developing immunogens that can effectively neutralize LASV.

Composition analysis of LASV GPC glycosylation modification
Picture composition analysis and structure spectrum of LASV GPC N-sugar

Simple and efficient whole-process sugar analysis program

The 2020 edition of the Chinese Pharmacopoeia newly added the “Mab N Glycograph Assay”, with the <Ultra Performance Liquid Chromatography-Hydrophilic Interaction Chromatography> as the first method. UPLC H-Class/FLR can provide high sensitivity in sugar analysis And resolution. Waters’ dedicated Glycan chromatographic column, coupled with the rapid sample preparation kit Glycoworks RapiFluor MS, Andrew+ intelligent pipetting robot and SmartMS-enabled BioAccord system, provides a full set of configuration and service programs for the workflow to ensure that the analysis method is ready to run. Follow-up can be transferred to QC and routine testing laboratories without any worries.


▐ Improved polysaccharide characterization method: Waters HILIC-UPLC chromatographic method combined with NIBRT robust online polysaccharide database

Waters and the National Institute of Biotechnology Research and Training (NIBRT) in Ireland jointly developed a comprehensive solution for polysaccharide analysis. The solution is based on the advanced GlycoBase 3+ database and ACQUITY UPLC system, as well as HILIC-UPLC separation columns and chemicals optimized for polysaccharide analysis.

National Institute of Biotechnology Research and Training (NIBRT)
GlycoBase 3+ is a non-public database that can be queried through the Internet. The database contains standard retention time information, expressed in glucose units or GU values, with more than 600 labeled N-sugar structures. All data are obtained by systematic analysis of free polysaccharides in a large number of glycoproteins using Waters UPLC technology and NIBRT polysaccharide analysis platform. It provides powerful tools for researchers engaged in the design, development and optimization of biological process in the production of biopharmaceuticals, biopharmaceutical quality research and control workers, cancer and other disease biomarker kits and the internal mechanism of glycosylation process.


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