Structural analysis of sulfated N-linked oligosaccharides in erythropoietin.
We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by (1)H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.
Kawasaki
N
N
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501 Japan.
Haishima
Y
Y
Ohta
M
M
Itoh
S
S
Hyuga
M
M
Hyuga
S
S
Hayakawa
T
T
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D002853:Chromatography, Liquid; D006224:Cricetinae; D004921:Erythropoietin / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization; D013402:Sugar Alcohols / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry*
D009844:Oligosaccharides; D011994:Recombinant Proteins; D013402:Sugar Alcohols; D004921:Erythropoietin; D013455:Sulfur; D006026:Glycoside Hydrolases