title: Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin.
abstract: Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR-AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N24, N38, N83, and O126 were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR-AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins.
authors: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
authors: Jing
authors: J
authors: Jiang
authors: Fang
authors: F
authors: Tian
authors: Yun
authors: Y
authors: Cai
authors: Xiaohong
authors: X
authors: Qian
authors: Catherine E
authors: CE
authors: Costello
authors: Wantao
authors: W
authors: Ying
mesh_terms: D002853:Chromatography, Liquid; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin