Analysis of methylated O-glycan alditols by reversed-phase NanoLC coupled CAD-ESI mass spectrometry

Abstract

The structural diversity of mucin-type O-glycans (O-GalNAc core-based) exceeds that of N-linked glycans by far. Structural analysis of this type of protein modification is hampered, however, by the unavailability of specific endo-acetylgalactosaminidases that would cleave the intact oligosaccharide from threonine or serine residues. Chemical cleavage is either performed by hydrazinolysis resulting in reducing glycans ready for terminal labeling reactions or by the classical reductive β-elimination, which yields the chemically inert alditols. Composition and sequence of the O-glycans can be analyzed by mass spectrometry using FAB-, MALDI-, or ESI-ionization technology. A significant increase in sensitivity and a facilitated fragmentation of the sugar chains can be achieved by methylation of free hydroxyl groups according to a modified procedure based on NaOH/DMSO and methyl iodide. Permethylated glycan alditols are readily separated on reversed-phase capillary columns under conditions similar to those used in peptide chromatography, and their sequences can be deduced from MS/MS spectra registered after collision-induced fragmentation of parental proton or sodium adduct ions using a Q-TOF mass spectrometer. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.