Characterization of sugars and glycans from ovalbumin using a combination of derivatization, high-performance liquid chromatography and mass spectrometry

Abstract

A combination of derivatization, reversed-phase high performance liquid chromatography (RP-HPLC), and mass spectrometry techniques has been explored for the characterization of small sugars, medium-size oligosaccharide , and glycans detached from chicken egg albumin (ovalbumin) by hydrazinolysis. The PMP derivatization method was preferred over pyridylamination owing to the simplicity of the reaction involved and, most importantly, its better enhancement of ionization efficiency in fast atom bombardment (FAB), electrospray (ESI), and matrix-assisted laser desorption/ionization (MALDI-MS). For oligosaccharides with more than three rings, only PMP-derivatives produced structurally related information, and only ESI and MALDI were utilized while FAB was dropped. Sensitivity studies were carried out using a standard oligosaccharide, tetraglucose. The other aim was to study the fragmentation patterns of PMP-derivatives. Qualitative fragmentation studies of PMP-derivatives were also carried out with typical N-linked oligosaccharides obtained commercially. PMP-derivatives of these standard oligosaccharides not only produce abundant molecular ions, but also informative sequence-related fragment ions in ESI-MS and ESI-MS/MS while the MALDI mass spectra mainly yielded molecular ions, such as (M + H) $\sp+,$ and (M + NA) $\sp+.$ With this information in hand, the derivatization method was then applied to N-glycans cleaved from ovalbumin, which has only one N-glycosylation site. (Abstract shortened by UMI.)