Dataset Information

An integrated strategy reveals complex glycosylation of erythropoi-etin using top-down and bottom-up mass spectrometry

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Yudong Guan

LAB HEAD: Hartmut Schlüter

PROVIDER: PXD024835 | Pride | 2021-06-14

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	EPO-3_chymotrypsin_glycopeptide_1.raw	Raw
	EPO-3_chymotrypsin_glycopeptide_2.raw	Raw
	EPO-3_chymotrypsin_glycopeptide_3.raw	Raw
	EPO-3_chymotrypsin_peptide_1.csv	Csv
	EPO-3_chymotrypsin_peptide_1.ms1	Other

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Publications

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry.

Guan Yudong Y Zhang Min M Gaikwad Manasi M Voss Hannah H Fazel Ramin R Ansari Samira S Shen Huali H Wang Jigang J Schlüter Hartmut H

Journal of proteome research 20210610 7

The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed 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 identification. Permethylated N-glycans, peptides, and en ...[more]

PMID: 34110173

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