Resolution of oligosaccharides in glycopeptides using immobilized Endo-M and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

The resolution of asparagine-linked oligosaccharides in glycopeptides was carried out by combination of the transglycosylation reaction and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The resolution of the oligosaccharides is based on the enzymic transglycosylation reaction with Endo-beta-N-acetylglucosaminidase (Endo-M) isolated from Mucor hiemalis. The oligosaccharides were transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M to produce the fluorescent oligosaccharides. In the present research, the enzyme was also immobilized in the well of a microassay plate by the sol-gel technique. The transglycosylation reaction was easily managed due to the immobilization. Furthermore, multiple use was possible by the encapsulated Endo-M. The resulting fluorescent oligosaccharides were separated by UPLC and efficiently detected by ESI-TOF-MS. Several oligosaccharides in ovalbumin were successfully identified by the proposed procedure.