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.
Keywords: CID; HCD; N-glycan; O-glycan; VWF; glycosylation; mass spectrometry; microheterogeneity; occupancy; plasma von Willebrand Factor.