Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Guan Y

PROVIDER: S-EPMC9472269 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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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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Dataset Information

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

Publications

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

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