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 fucose-containing oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by high-performance liquid chromatography (HPLC) on normal-phase and reversed-phase columns, and by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Six out of eight PA-oligosaccharides were clearly separated by both normal- and reversed-phase HPLC at a column temperature of 40 degrees C, but two PA-oligosaccharides, lacto-N-fucopentaose II [Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4GIcPA] and lacto-N-fucopentaose III [Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4GIcPA], were not separated. The two unresolved PA-oligosaccharides were finally separated by reversed-phase HPLC at a column temperature of 11 degrees C. MALDI-TOF mass spectra of PA-oligosaccharides demonstrated pseudo-molecular ions as the predominant signals, therefore information about the molecular mass of each PA-oligosaccharide was easily obtained. Post-source decay (PSD) MALDI-TOF mass spectra of PA-oligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PA-oligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PA-oligosaccharides by HPLC, MALDI-TOF mass spectrometry, and PSD MALDI-TOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDI-TOF mass spectrometry or HPLC and PSD MALDI-TOF mass spectrometry is quite useful for the structural characterization of sub-pmol or pmol levels of fucose-containing oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.
Suzuki
M
M
Sphingolipid Expression Laboratory, RIKEN Frontier Research System, Wako-shi, Saitama, Japan.
Suzuki
A
A
D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005643:Fucose / Q000737:chemistry*; D006028:Glycosphingolipids / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014312:Trisaccharides / Q000032:analysis / Q000737:chemistry
C433829:GalNAc-1-3-(Fuc-1-2)Gal-1-3-GlcNAc-1-3-Gal-1-4-Glc-PA; C433830:GalNAc-1-3-(Fuc-1-2)Gal-1-4-Glc-PA; D006028:Glycosphingolipids; C024050:Le(b)-active glycolipid; D009844:Oligosaccharides; D014312:Trisaccharides; D005643:Fucose; C045442:lacto-N-fucopentaose I; C031420:2'-fucosyllactose