earticle

영어

Recombinant coagulation factor IX (rFIX) is an extremely heterogeneous glycoprotein having multiple N-and O-glycosylation which influence on pharmacological functions including biological activity and in vivo stability. The study of glycosylation on rFIX has mainly focused on N-glycans due to an inherent complexity of O-glycosylation and the absence of analytical platform. Here, we comprehensively characterized the O-glycosylation on recombinant coagulation factor IX (rFIX) using MS-based glycomic and glycoproteomic approach. Briefly, O-glycans were chemically released using β-elimination while O-glycopeptides were prepared by pronase digestion to determine site-specific O-glycosylation distribution. Successfully we could identify unique O-glycosylation modifications such as O-fucosylation and O-glucosylation as well as a typical O-glycosylation on rFIX using nanoLC/MS and Tandem MS. Particularly, through O-glycopeptide analysis, we demonstrated that O-glucosylation and O-fucosylation were uniformly displayed on O-sites Serine 53 and 61 of EGF domain, respectively. While core 1-type O-glycosylations were overwhelmingly distributed across other O-sites (Threonine 159, 169, 172, and 179) in activation peptide of rFIX. Site-specific profiling including micro-and macro-heterogeneity could provide macroscopic aspect to figure out O-glycosylation pattern decorated on rFIX.