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Glycobiology Advance Access published online on December 22, 2004
Glycobiology, doi:10.1093/glycob/cwi033
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© Oxford University Press 2004; all rights reserved.
Received June 28, 2004
Revised November 24, 2004
Accepted December 16, 2004

Article

Site-specific glycosylation analysis of human apolipoprotein B100 using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Akira Harazono 1*, Nana Kawasaki 1, Toru Kawanishi 1, and Takao Hayakawa 1

1 National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, 1-18-1 Kami-yoga, Setagaya-Ku, Tokyo 158-8501, Japan

* To whom correspondence should be addressed.
Akira Harazono, E-mail: harazono{at}nihs.go.jp


  Abstract

Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). ApoB100 was reduced and carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI/MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions such as m/z 204 (HexNAc) and 366 (HexHexNAc) in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides while Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210 and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn 3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides including high-mannose, hybrid and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding.

Keywords: apolipoprotein B100/N-linked oligosaccharide/glycopeptide/liquid chromatography electrospray mass spectrometry/product ion spectrum.
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