PXD055221

PXD055221 is an original dataset announced via ProteomeXchange.

Title	Decoding protein glycosylation by an integrative mass spectrometry-based de novo sequencing strategy
Description	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.
HostingRepository	iProX
AnnounceDate	2024-08-24
AnnouncementXML	Submission_2025-03-11_19:49:15.083.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Jing Gao
SpeciesList	scientific name: Bos taurus; NCBI TaxID: 9913; scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive; Orbitrap Eclipse; Q Exactive HF; Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2024-08-27 01:19:10	ID requested
⏵ 1	2025-03-11 19:49:15	announced

Gao J, Chen H, Yin H, Chen X, Yang Z, Wang Y, Wu J, Tian Y, Shao H, Wen L, Zhou H, Sequencing Strategy. JACS Au, 5(2):702-713(2025) [pubmed]

submitter keyword: glycoprotein, protein de novo sequencing, mass spectrometry, glycosylation characterization

Zhou Hu
contact affiliation	Shanghai Institute of Materia Medica, Chinese Academy of Sciences
contact email	zhouhu@simm.ac.cn
lab head
Jing Gao
contact affiliation	Shanghai Institute of Materia Medica, Chinese Academy of Sciences
contact email	jinggao@simm.ac.cn
dataset submitter

iProX dataset URI