title: A two-dimensional array for simultaneous sequencing of N- and o-glycans and their glycoforms on specific glycosylation sites.
abstract: Glycans play major roles in living organisms. Thus, essential information is required on diverse glycans, their location, and moieties in proteins, as well as for technology in a high-throughput manner, for improving functional glycomics. In the present study, we describe a new approach involving a 2-D array, which has the potential to fulfill both requirements. The first dimension of the array is composed of various lectins immobilized to a MALDI plate. The second dimension consists of initial proteolysis, then sequential exoglycosidase digestion using highly specific enzymes. The products of such digestions are peptide/glycopeptide mixtures conjugating different glycan fragments from which the exoglycosidase has removed specific terminal residues. Consequently, a series of spectra are obtained when lectin-attached products are analyzed by MALDI-TOF MS. By using well-known glycoproteins and NKp46D2-Ig, a recombinant fusion natural killer receptor with unknown glycans produced in CHO cells, we proved the usefulness of the method, demonstrating rapid and simultaneous determination of N- and O-glycan sequences, their glycan moieties, and subtypes on each of the determined glycosylation sites. This strategy provides a tool that can rapidly explore glycan structures and might contribute to a better understanding of process- and disease-related glycoproteins.
authors.affiliation: Department of Biotechnology Engineering, Faculty of Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Yaron
authors.initials: Y
authors.lastname: Tzur
authors.forename: Amir
authors.initials: A
authors.lastname: Markovich
authors.forename: Rachel G
authors.initials: RG
authors.lastname: Lichtenstein
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011134:Polysaccharides