Estimation of the number of O-linked oligosaccharides per heavy chain of human serum IgA1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the hinge glycopeptide.
In our previous study, gas-phase hydrazinolysis was used to analyze the glycoform of the O-linked oligosaccharide of human serum IgA1. All O-linked oligosaccharide chains are known to be present in the hinge portion. However, the number of O-linked oligosaccharide chains on IgA1 remained unclear. In order to determine the number of linked sugar chains, we applied matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to the hinge glycopeptide prepared from human serum IgA1. MALDI-TOFMS did not show clear peaks, probably due to the microheterogeneity of the structure of each sugar chain. However, elimination of peripheral sialic acid and galactose residues by sequential treatment with neuraminidase and beta-galactosidase gave clear mass spectra with several sharp peaks. On the basis of these spectra, we conclude that IgA1 prepared from normal human serum carries different numbers of sugar chains. There are two major populations, one contains five GalNAc residues and the other four GalNAc residues. On the other hand, the hinge glycopeptide prepared from myeloma IgA1 was composed mainly of one population containing four GalNAc residues. Earlier, we reported incomplete glycosylation of IgA1 isolated from the serum of an IgA1 myeloma patient. In this experiment, the presence of four O-linked oligosaccharides per heavy chain of IgA1 from a myeloma patient was found. The reason why only four out of five sites on the hinge glycopeptide were fully glycosylated in the IgA1 from the IgA1 myeloma patient is not clear.
Department of Biochemistry and Medicine, School of Medicine and Nursing, Kitasato University, Kanagawa.
H
H
Iwase
A
A
Tanaka
Y
Y
Hiki
T
T
Kokubo
I
I
Ishii-Karakasa
Y
Y
Kobayashi
K
K
Hotta
D000595:Amino Acid Sequence; D006020:Glycopeptides / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood / Q000737:chemistry* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009101:Multiple Myeloma / Q000276:immunology; D009844:Oligosaccharides / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D006020:Glycopeptides; D007070:Immunoglobulin A; D007143:Immunoglobulin Heavy Chains; D009844:Oligosaccharides