Mutual separation of hinge-glycopeptide isomers bearing five N-acetylgalactosamine residues from normal human serum immunoglobulin A1 by capillary electrophoresis

Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Gal beta 1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and beta-ga lactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obt ained. The HGP-SG was separated into three major peaks, A, B and C, by high -performance liquid chromatography (HPLC). Each glycopeptide fraction was f urther separated by capillary electrophoresis (CE). Peaks A, B and C with H PLC abundantly contained HP33 bearing five and six N-acetylgalactosamine re sidues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among thes e glycopeptide peaks, only the HGP33-5GN peak was partly split into two pea ks based on the CE analysis - HGP33-5GN-alpha and -beta. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well sep arated into the alpha and beta forms try CE analysis. No differences in the ir mass and peptide portion were observed between HGP25-5GN-alpha and -beta . Therefore, the obtained result might indicate that HGP25-5GN-alpha was an isomer of HGP25-5GN-beta differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue. (C) 1999 Elsevier Science B.V. All rights reserved.