Decoding Protein Glycosylation by an Integrative Mass Spectrometry-Based De Novo Sequencing Strategy
peer-reviewed · JACS Au · 2025
| Date | 2025-01-22 |
| Type | peer-reviewed |
| Venue | JACS Au |
| Publisher | American Chemical Society (ACS) |
| Contribution | downstream-application |
| DOI | 10.1021/jacsau.4c00960 |
Abstract
Glycoproteins, representing more than 50% of human proteins and most biopharmaceuticals, are crucial for regulating various biological processes. The complexity of multiple glycosylation sites often leads to incomplete sequence coverage and ambiguous glycan modification profiles. Here, we developed an integrative mass spectrometry-based approach for decoding unknown glycoproteins, which is featured with the combination of deglycosylation-mediated de novo sequencing with glycosylation site characterization. We utilized the enzymatic deglycosylation of N- / O- glycans to achieve comprehensive sequence coverage. Additionally, EThcD fragmentation enables the identification of high-quality long peptides, facilitating precise protein assembly. We subsequently applied this method to de novo sequencing of the highly glycosylated therapeutic fusion protein Etanercept (Enbrel). We also sequenced three new tumor necrosis factor receptor:Fc-fusion biologics with largely unknown sequences, unveiling subtle distinctions in the primary sequences. Furthermore, we characterized N- and O- glycosylation modifications of these proteins at subunit, glycopeptide, and glycan levels. This strategy bridges the gap between the de novo sequencing and glycosylation modification, providing comprehensive information on the primary structure and glycosylation modifications for glycoproteins. Notably, our method could be a robust solution for accurate sequencing of the glycoproteins and has practical value not only in basic research but also in the biopharmaceutical industry.
Methods and tools
- Deglycosylation-assisted glycoprotein de novo sequencing: Integrative strategy for glycoprotein primary structure: enzymatic N-/O- deglycosylation to open up sequence coverage, EThcD to yield long peptides, PEAKS AB de novo sequencing to assemble the backbone, then glycosylation site and glycan characterisation on top. Demonstrated on Etanercept and three TNFR:Fc-fusion biologics of unknown sequence.
- PEAKS: Commercial DP-based de novo