PXD024835

PXD024835 is an original dataset announced via ProteomeXchange.

Title	An integrated strategy reveals complex glycosylation of erythropoi-etin using top-down and bottom-up mass spectrometry
Description	The characterization of therapeutic glycoproteins is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis using first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identi-fication. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.
HostingRepository	PRIDE
AnnounceDate	2021-09-14
AnnouncementXML	Submission_2021-09-14_08:19:46.837.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Yudong Guan
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2021-03-18 07:39:35	ID requested
⏵ 1	2021-09-14 08:19:47	announced

Guan Y, Zhang M, Gaikwad M, Voss H, Fazel R, Ansari S, Shen H, Wang J, Schl, ü, ter H, An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry. J Proteome Res, 20(7):3654-3663(2021) [pubmed]

submitter keyword: Glycosylation, Mass spectrometry, Python scripts, Top-down, Bottom-up, Erythropoietin

Hartmut Schlüter
contact affiliation	Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany
contact email	hschluet@uke.de
lab head
Yudong Guan
contact affiliation	Southern University of Science and Technology
contact email	guan181992@163.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD024835
     1. Label: PRIDE project
     2. Name: An integrated strategy reveals complex glycosylation of erythropoi-etin using top-down and bottom-up mass spectrometry