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 C18 column eluted in acetonitrile-1 mM 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, 0126, 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.
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan. ohta@nihs.go.jp
M
M
Ohta
N
N
Kawasaki
S
S
Hyuga
M
M
Hyuga
T
T
Hayakawa
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry*; D010449:Peptide Mapping; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D006020:Glycopeptides; D004921:Erythropoietin