Structural Characterization of Fucose-Containing Oligosaccharides by High-Performance Liquid Chromatography and Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry

Eight pyridylamino (PA) derivatives of fucosecontaining oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by highperformance liquid chromatography (HPLC) on normalphase and reversedphase columns, and by matrixassisted laser desorption/ionization timeofflight MALDITOF) mass spectrometry. Six out of eight PAoligosaccharides were clearly separated by both normal and reversedphase HPLC at a column temperature of 40 C, but two PAoligosaccharides, lactoNfucopentaose II [Gal?1-3(Fuc?1-4)GlcNAc?1- 3Gal?1-4GlcPA] and lactoNfucopentaose III Gal?1-4(Fuc?1-3)GlcNAc?1-3Gal?1-4GlcPA], were not separated. The two unresolved PAoligosaccharides were finally separated by reversedphase HPLC at a column temperature of 11 C. MALDITOF mass spectra of PAoligosaccharides demonstrated pseudomolecular ions as the predominant signals, therefore information about the molecular mass of each PAoligosaccharide was easily obtained. Postsource decay (PSD) MALDITOF mass spectra of PAoligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PAoligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PAoligosaccharides by HPLC, MALDITOF mass spectrometry, and PSD MALDITOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDITOF mass spectrometry or HPLC and PSD MALDITOF mass spectrometry is quite useful for the structural characterization of subpmol or pmol levels of fucosecontaining oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.