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 H-1-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.