No abstract

Human coagulation factor VIII (FVIII) is a key co-factor in the clotting cascade, the deficiency of which leads to Hemophilia A. Human plasma-derived (pdFVIII) and recombinant FVIII (rFVIII) had been used as effective products to prevent and treat bleeding episodes. Both FVIII products share identical amino acid sequences and appear to be equivalent as of clinical efficiency. However, systemic reviews found an increased risk of neutralizing antibody (or inhibitor) development with recombinant products. FVIII is a highly glycosylated protein, and its glycosylation pattern is specific to host cells and environments. The roles of glycosylation in immune responses toward pdFVIII and rFVIII are yet to be defined. Herein, we systemically profiled N-and O-glycomes of pdFVIII and rFVIII using a mass spectrometrybased glycoproteomic strategy. A total of 110 site-specific N-glycopeptides consisting of 61 N-glycoforms were identified quantitatively from rFVIII and pdFVIII. Additionally, 31 O-glycoforms were identified on 23 peptides from rFVIII and pdFVIII. A comprehensive comparison of their site-specific glycan profiles revealed distinct differences between the glycosylation of pdFVIII and rFVIII.

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.