Mass Spectrometry Based Analysis of Protein N-Glycosylation in Biomarker Discovery and Gene Therapy with the Study Models-Hemophilia A Inhibitor Development and rAAV

Protein glycosylation is one of the critical post-translational modifications (PTMs) and practically engaged with a wide range of physiological and biological processes. Glycosylation is the most dynamic post-translational modification and an individual\u27s glycome changes overcome the genetic factors and get affected by environmental factors which eventually reflect his lifestyle, physiological conditions and wellbeing. The flow study, we attempted to add this information to comprehend the glycoprotein biomarker identified with inhibitor advancement and connected the glycosylation related changes to the biochemical pathway of inhibitor development against rFVIII in HA population. We performed the study with mice and human models. Plasma and IgG N-glycome examination is one of the important methodologies to identify the biomarker related to numerous conditions. The N-glycome pattern also varies in response to the treatment. The treatment-related modifications also reaffirm the observations noted in the progression of the disease. Similarly, the glycosylation can be a useful strategy to modify the protein-based drugs to enhance its mode of action. The variant of AAV can be a potential capsid engineering technique to alter the tropism and improve the gene delivery range of host cell range for engineering a better gene delivery system. The small amount of and glycan variants are difficult to detect in a complex biological mixture, which may require various enrichment strategies, and sample preparations help to enhance the detection sensitivity in mass spectrometry. Due to the with the development of state-of-the-art mass spectrometry (MS) technology, we tried to identify N-glycan biomarkers related to inhibitor development in HA. Also, we decided to study the response of the patient after emicizumab. Additionally, we identified N-glycosylation in rAAV-8, which can be a potential direction for future capsid engineering

Site-Specific N-Glycosylation on the AAV8 Capsid Protein

Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs

An Insight into Glyco-Microheterogeneity of Plasma von Willebrand Factor by Mass Spectrometry

Human plasma von Willebrand Factor (VWF) plays essential roles in primary hemostasis in cooperation with other coagulations factors. There is ample indication that glycosylation affects many biological phases during the protein life cycle. However, comprehensive characterization of all probable N-glycosites simultaneous with O-glycosites is still not fully revealed. Thus, the intention of this exploration was to estimate the occupancy of all canonical N-glycosites besides simultaneous characterization of N- and O-glycoforms. An RP–LC–MS/MS system functionalized with CID and HCD tandem mass was utilized to analyze VWF. N-Glycosite occupancy varied along the protein backbone chain. Out of 257 HCD spectra, 181 characterized glycoforms were specified as either N- or O-glycosites. Sequential cleavage of glycosidic bonds along with Human Database mass matching have confirmed the glycoform structures. A total of 173 glycoforms represented most commonly biantennary and infrequently tri- and tetra-antennary N-glycans beside high mannose, hybrid, ABH antigen-terminated, and sulfated N-glycans. Many glycoforms were common across all N-sites. Noteworthy, previously unreported N-glycosites within domain D′(TIL′-E′) showed glycosylation. Moreover, sialylated core 1 and core 2 O-glycans were detected on 2298T. Given subtle characterization of site-specific glycoforms, we can attain a profound understanding of the biological roles of VWF as well as facilitate the production of VWF-based therapeutics