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

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...

Full description

Saved in:
Bibliographic Details
Published inJournal 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
LanguageEnglish
Published Amsterdam Elsevier B.V 23.02.2001
Elsevier
Subjects
Online AccessGet full text

Cover

Loading…
More Information
Summary: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.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0021-9673
DOI:10.1016/S0021-9673(00)01116-X