In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MS(n)) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + H]+ ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + H]+ ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MS(n) analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MS(n) analysis, efficiently characterized the structures of beta2-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.

Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.

It was found in our previous studies that the concentration of fucosylated haptoglobin had increased in the sera of patients with pancreatic cancer (PC) compared to those of other types of cancer and normal controls. Haptoglobin, an acute phase protein, has four potential N-glycosylation sites, although it remains unknown which site is responsible for the change in fucosylated N-glycans. In the present study, site-specific N-glycan structures of haptoglobin in sera obtained from patients with PC or chronic pancreatitis (CP) were analyzed using liquid chromatography-electrospray ionization mass spectrometry. Mass spectrometry analyses demonstrated that concentrations of total fucosylated di-, tri-and tetra-branched glycans of haptoglobin increased in the sera of PC patients. Tri-antennary N-glycans containing a Lewis X-type fucose markedly increased at the Asn211 site of haptoglobin Nglycans. While fucosylated N-glycans derived from serum haptoglobin of patients with CP slightly increased, di-fucosylated tetraantennary N-glycans were observed only at this site in PC patients, and were absent in the haptoglobin of normal controls and individuals with CP. Thus, the present study provides evidence that site-specific analyses of N-glycans may be useful as novel tumor markers for PC.

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.

The oligosaccharide structures of prostate specific antigen (PSA) are expected to be useful in discriminating prostate cancer from benign conditions both accompanied by increased serum PSA levels. A large proportion of PSA forms a covalent complex with a glycoprotein, alpha(1)-antichymotrypsin, in human blood. In the present study, the glycan profiles of free and complexed forms of PSA from cancer patient serum and of seminal plasma PSA were compared by analyzing the glycopeptides obtained by lysylendopeptidase digestion of the electrophoretically separated PSA with mass spectrometry. The profiles of the PSA N-glycans from the free and complexed molecules were quite similar to each other and consisted of fucosylated biantennary oligosaccharides as the major class. They were mostly sialylated, and a considerable sialic acid fraction was alpha2,3-linked as determined by Streptococcus pneumoniae neuraminidase digestion of the glycopeptides. In the seminal plasma PSA, high-mannose and hybrid types of oligosaccharides were predominant, and the sialic acids attached to the latter as well as to biantennary oligosaccahrides were exclusively alpha2,6-linked because they were removed by Arthrobacter ureafaciens neuraminidase but resistant to S. pneumoniae neuraminidase. Complex-type oligosaccharides from other sources were found in the seminal plasma sample, indicating that analysis of released glycans carries a risk of being misleading. The results suggest that identification of alpha2,3-linked sialic acids on PSA potentially discriminates malignant from benign conditions, if the analysis is applied to oligosaccharides specifically attached to the N-glycosylation site of PSA in either a free or a complexed form in the serum.