Site-specific glycosylation analysis of human apolipoprotein B100 using LC/ESI MS/MS

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, 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, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, 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.

Exploring the Sialiome Using Titanium Dioxide Chromatography and Mass Spectrometry

“…Half of the eluate from the TiO 2 monosaccharides from the glycopeptide, indicating that the charge is predominantly retained on the peptide (Y-type fragments (23)). Three oxonium ions are present that are diagnostic for SA-containing glycans (m/z 274.07, 292.09, and 657.23 (38)). The mass of the peptide can be deduced when taking into account the common core structure of N-linked glycans, i.e.…”

Glycosyltransferases and non-alcoholic fatty liver disease

“…Apolipoprotein B (ApoB)-100, a large secretory glycoprotein with 4536 amino acid residues, is an important component of VLDL [66] . It has 19 potential glycosylation sites (Asn-X-Ser/Thr), and 16 of them have been reported to be glycosylated [67,68] .…”

Untitled

“…It can be applied to glycosylated peptides, and glycopeptide MS is a key interfacing technique in proteomics and functional glycomics. The increasing number of reports clearly indicates that the strategy is su‹ciently established to allow practical site-speciˆc characterization of the N-linked oligosaccharide proˆles of even highly heterogeneous glycoproteins (3,4). In contrast, this is not the case for the glycopeptide containing O-glycans.…”