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bioRxiv · 10.1101/2021.02.09.430553

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

Guan, Y.·Zhang, M.·Gaikwad, M.·Voss, H.·Fazel, R.·Ansari, S.·Shen, H.·Wang, J.·Hartmut, S.
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Abstract

The characterization of glycoproteins, like erythropoietin, is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis of a 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 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). 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.

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Guan, Y., Zhang, M., Gaikwad, M., Voss, H., Fazel, R., Ansari, S., Shen, H., Wang, J., Hartmut, S.. 2021-02-10. An integrated strategy reveals complex glycosylation of erythropoietin using top-down and bottom-up mass spectrometry. https://doi.org/10.1101/2021.02.09.430553

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