Improved Method Supporting Maldi-Ms Analysis of Sialyloligosaccharides Including Their Structural Isomers

Abstract

Structural and functional analyses of glycans in glycoconjugates have become an essential study for understanding various biological events. Mass spectrometry (MS) has been developed as one of the main analytical methods in glycan study. However, the analysis of isomers of glycans has been an important subject accompanying on the MS analysis. We reported a fluorescent labeling of carboxy group of sialic acid by amidation with 2-(2-pyridylamino) ethylamine (PAEA). The method suppressed the dissociation of sialic acid in MALDI-MS (Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry) and enabled both stable MALDI-MS analysis and high sensitive chromatographic analysis of sialyloligosaccharides and gangliosides. TLC (Thin Layer Chromatography) and HPLC (High Performance Liquid Chromatography) analyses provided important informations on glycosidic linkages of sialic acids that could not be obtained by MS analysis alone. High sensitive detection of the chromatographic separation improved the discrimination and the purification of each component of the isomers. In this study, the PAEA-amidation of sialyloligosaccharide was checked by TLC. The reaction mixture developed on a TLC plate was transferred to PVDF (polyvinylidene difluoride) membrane and the spot corresponding to the PAEA-sialyloligosaccharide was cut off. The content was extracted and then submitted to MALDI-TOF-MS analysis after one step treatment using a reversed-phase cartridge, resulted in providing the reliable mass spectrum. As described above, our improvement simplified the process from the detection on chromatography to the purification of PAEA-sialyloligosaccharides for MALDI-MS analysis and enabled detailed sialyloligosaccharide structural analysis including the discrimination of isomers.