An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry

Guan，Yudong1; Zhang，Min2; Gaikwad，Manasi2; Voss，Hannah2; Fazel，Ramin3; Ansari，Samira4; Shen，Huali5; Wang，Jigang1; Schlüter，Hartmut2

2021

10.1021/acs.jproteome.1c00221

JOURNAL OF PROTEOME RESEARCH   影响因子和分区

1535-3893

1535-3907

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.

bottom-up erythropoietin glycoinformatics glycosylation mass spectrometry Python scripts

SCI

英语

第一

WOS:000670662100024

BIOLOGY & BIOCHEMISTRY

2-s2.0-85108888431

Scopus

1.The First Affiliated Hospital (Shenzhen People's Hospital),Southern University of Science and Technology,Shenzhen,518055,China
2.Section Mass Spectrometric Proteomics,Institute of Clinical Chemistry and Laboratory Medicine,University Medical Center Hamburg-Eppendorf,Hamburg,20246,Germany
3.Reasearch and Innovation Center,Livogen Pharmed Co.,Tehran,1417755358,Iran
4.CinnaGen Medical Biotechnology Research Center,Alborz University of Medical Sciences,Karaj,3165933155,Iran
5.Department of Systems Biology for Medicine,School of Basic Medical Sciences,Fudan University,Shanghai,200032,China

Guan，Yudong,Zhang，Min,Gaikwad，Manasi,et al. An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry[J]. JOURNAL OF PROTEOME RESEARCH,2021.

Guan，Yudong.,Zhang，Min.,Gaikwad，Manasi.,Voss，Hannah.,Fazel，Ramin.,...&Schlüter，Hartmut.(2021).An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry.JOURNAL OF PROTEOME RESEARCH.

Guan，Yudong,et al."An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry".JOURNAL OF PROTEOME RESEARCH (2021).