Decoding protein glycosylation by an integrative mass spectrometry-based de novo sequencing strategy

ABSTRACT: Glycoprotein is one of the most complex biomacromolecule，accounting for over 50% of total human proteins and the majority of biopharmaceuticals, exerting profound influences on various essential biological processes. With multiple N-glycosylation sites and O- glycosylation sites on the protein backbone, decoding of unknown glycoprotein is very challenging due to the inherent variability and bias on the detectability of the glycopeptides, leading to incomplete coverage of certain backbone regions and ambiguous identification of glycan modifications. Here, we demonstrated an integrative approach for decoding glycoprotein, which is featured with combination of deglycosylation-mediated de novo sequencing with glycosylation site characterization. We utilized enzymatic deglycosylation for N-/ O- glycan to achieve complete sequence coverage, as well as EThcD fragmentation enabling the identification of high-quality long peptides to facilitate the precise protein assembly. We subsequently applied this method to de novo sequencing of Etanercept, a highly glycosylated therapeutic recombinant TNFR: Fc-fusion protein, and three new TNFR: Fc-fusion biologics whose sequence were largely unknown, unveiling subtle distinctions in the primary sequences. Finally, the N-/ O-glycosylation modifications of these proteins were characterized at different levels—subunit, glycopeptide, and glycan. We believe that this strategy bridges the gap between the de novo sequencing and glycosylation modification, providing the complete information of the primary structure and glycosylation modifications for glycoproteins. Notably, our method could be a robust solution for accurate sequencing the glycoproteins and has practical value in biopharmaceutical industry.

ORGANISM(S): Homo Sapiens Bos Taurus

SUBMITTER: Zhou Hu  

PROVIDER: PXD055221 | iProX | Sat Aug 24 00:00:00 BST 2024

REPOSITORIES: iProX