An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry

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 spectrome...

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Bibliographic Details
Published in:Journal of proteome research Vol. 20; no. 7; p. 3654
Main Authors: Guan, Yudong, Zhang, Min, Gaikwad, Manasi, Voss, Hannah, Fazel, Ramin, Ansari, Samira, Shen, Huali, Wang, Jigang, Schlüter, Hartmut
Format: Journal Article
Language:English
Published: United States 02.07.2021
Subjects:
Chromatography, Liquid
Erythropoietin
Glycopeptides
Glycosylation
Polysaccharides
Tandem Mass Spectrometry
glycosylation
mass spectrometry
Python scripts
glycoinformatics
bottom-up
erythropoietin
ISSN:1535-3907, 1535-3907
Online Access:Get more information
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Summary: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 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). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.
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ISSN:1535-3907
1535-3907
DOI:10.1021/acs.jproteome.1c00221