A Two-Dimensional Array for Simultaneous Sequencing of N- And O-Glycans and Their Glycoforms on Specific Glycosylation Sites

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-lg, 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.