Selective glycopeptide mapping of erythropoietin by on-line high-performance liquid chromatography–electrospray ionization mass spectrometry

• Published in: Journal of Chromatography A Vol. 910; no. 1; pp. 1 - 11
• Main Authors: Ohta, Miyako, Kawasaki, Nana, Hyuga, Sumiko, Hyuga, Masashi, Hayakawa, Takao
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 23.02.2001; Elsevier
• Subjects: Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Erythropoietin; Erythropoietin - chemistry; Fundamental and applied biological sciences. Psychology; Glycopeptides; Glycopeptides - chemistry; Glycoproteins; Peptide Mapping; Peptides; Proteins; Spectrometry, Mass, Electrospray Ionization - methods; Proteins; Glycoproteins; Erythropoietin; Peptides; Glycopeptides; Human; Molecular form; On line; Coupled method; Oligosaccharide; HPLC chromatography; Glycoprotein hormone; Electrospray; Heterogeneity; Analysis method; Glycopeptide; Recombinant protein; Mass spectrometry; Structural analysis; Microheterogeneity
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(00)01116-X

Selective glycopeptide mapping of recombinant human erythropoietin (rhEPO) used as a model glycoprotein was successfully carried out by on-line high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) using a Vydac C 18 column eluted in acetonitrile–1 m M ammonium acetate, pH 6.8. rhEPO expressed in a Chinese hamster ovary clone was exhaustively digested into four glycopeptides and nine peptides with endoproteinase Glu-C. Both glycopeptides and peptides were eluted with trifluoroacetic acid as the eluent, whereas only glycopeptides were eluted selectively with ammonium acetate in the following order: N38, N24, O126, and N83. Furthermore, many glycoforms included in each glycopeptide were found to be separated by differences in the numbers of sialic acid and N-acetyllactosaminyl repeats. Twenty, 16 and 22 different N-linked oligosaccharides were determined at Asn24, 38, and 83, respectively, and two different O-linked oligosaccharides were observed at Ser126. Our method is simple, rapid, and useful for determining the carbohydrate structures at each glycosylation site and for elucidating the site-specific carbohydrate heterogeneity.