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 Oglycan 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.

Multiomic profiling of medulloblastoma reveals subtype-specific targetable alterations at the proteome and N-glycan level

“…MaxQuant were used for extracting all the detected masses and m/z from MS raw data of permethylated reducing N-glycans. Home-made Python-scripts is used to extract and calculate monosaccharide compositions based on the molecular weight of each derivatized N-glycan 83 . The N-glycan structures were identified manually based on full MS, m/z and MS2 ion according to Xcalibur and Glycoworkbench 2.1.…”

Comprehensive Glycosylation Characterization of Recombinant Human Erythropoietin by Electron-Activated Dissociation Mass Spectrometry

“…Depending on the linkage between glycans and protein in therapeutic protein, glycosylation is divided into N-glycosylation and Oglycosylation [4]. At present, the main methods of glycosylation analysis are liquid chromatography (LC) [5], liquid chromatography-mass spectrometry (LC-MS) [6][7], capillary electrophoresis (CE) [8] and capillary electrophoresis-mass spectrometry (CE-MS) [9][10]. Among them, LC-MS method based on high resolution mass spectrometry gains an important attention owing to its high sensitivity, high resolution and high accuracy.…”