Differential analysis of site-specific glycans on plasma and cellular fibronectins: application of a hydrophilic affinity method for glycopeptide enrichment

Abstract

Isolation of glycopeptides utilizing hydrogen bonding between glycopeptide glycans and a carbohydrate-gel matrix in the organic phase is useful for site-specific characterization of oligosaccharides of glycoproteins, when combined with mass spectrometry. In this study, recovery of glycopeptides was improved by including divalent cations or increasing the organic solvent in the binding solution, without losing specificity, whereas it was still less effective for those with a long peptide backbone exceeding 50 amino acid residues. The method was then applied to the analysis of glycan heterogeneities at seven N-glycosylation sites in each of the plasma and cellular fibronectins (FNs). There was a remarkable site-specific difference in fucosylation between these isoforms; Asn1244 selectively escaped the global fucosylation of cellular FN, whereas only Asn1007 and Asn2108 of the plasma isoform underwent modification. In addition, a new O-glycosylation site was identified at Thr279 in the connecting segment between the fibrin- and heparin-binding domain and the collagen-binding domain, and the glycopeptide was reactive to a peanut agglutinin lectin. Considering that another mucin-type O-glycosylation site lies within a different connecting segment, the O-glycosylation of FN was suggested to play a significant role in segregating the neighboring domains and thus maintaining the topology of FN and the domain functions. In addition, the method was applied to apolipoprotein B-100 (apoB100) whose N-glycan structures at 17 of 19 potential sites have been reported, and characterized the remaining sites. The results also demonstrated that the enriched glycopeptide provides resources for site-specific analysis of oligosaccharides in glycoproteomics.

Surfactant protein D suppresses lung cancer progression by downregulation of epidermal growth factor signaling

“…48 Amino acid sequencing was carried out by collision-induced dissociation (CID) and multiple-stage tandem MS using an LTQ-XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA). 49 The glycosylated peptide samples were dissolved in a 0.1% formic acid and 20% (v/v) methanol solution and directly infused by using a spray tip (New Objective, Woburn MA) for infusion mode nanoelectrospray ionization MS.…”

Molecular heterogeneity of gelatin-binding proteins from human seminal plasma

“…Concerning glycosylation, FN is reported to contain both N-and O-glycans and carbohydrates ac count for 5% of its molecular mass [7]. FN from various sources differs in the degree of sialylation and the type of linkage of sialic acid, as well as in the absence or presence of fucose [47,[49][50][51][52][53][54]. Thus, the N-glycans of plasma FN are complex-type biantennary oligosaccharides, largely sialylated with predominance of the sialic acid α2,6 Gal linkage, whereas fetal/ oncofetal FN, which is upregulated during development/ transformation, is core fucosylated and generally not or poorly sialylated, with predominance of the sialic acid α2,3 Gal linkage.…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…62 Polar glycopeptides are retained on the HILIC phase, while the hydrophobic peptides are washed off with high organic concentrations in the mobile phase, which allows the separation of glycosylated and non-glycosylated peptides. 119,120 Retained glycopeptides can then be eluted by increasing the water content of the mobile phase. Recently, tryptic glycopeptides have been finely separated on a 1.7-μm amide column, with elution in the order of non-glycosylated, O-glycosylated and N-glycosylated glycopeptides.…”