Hog or porcine
gastric mucin resembles the human source in carrying not only
blood group antigens but also the rather rare α4-GlcNAc-capped terminal
epitope functionally implicated in protection against Helicobacter pylori
infection. Being more readily available and reasonably well characterized, it serves as a good
reagent for immunobiological studies, as well as a standard for analytical methodology developments. Current approaches in mass spectrometry (MS)-based glycomic mapping remain vastly inadequate in revealing the full complexity of glycosylation, particularly for cases such as the extremely heterogeneous O-glycosylation of mucosal
mucins that can be further sulfated. We demonstrate here a novel concerted workflow that extends the conventional matrix-assisted
laser desorption/ionization–mass spectrometry (MALDI-MS) mapping of permethylated
glycans in positive ion mode to include a further step of sulfoglycomic analysis in negative ion mode. This was facilitated by introducing a mixed-mode solid-phase extraction step, which allows direct cleanup and simultaneous fractionation of the permethylated
glycans into separate non-sulfated and sulfated pools in one single step. By distinct MALDI-MS/MS fragmentation patterns, all previously known structural features of porcine
gastric mucin including the terminal
epitopes and location of
sulfates could be readily defined. We additionally showed that both arms of the core 2 structures could be extended via 6-O-sulfated GlcNAc to yield a series of disulfated O-
glycans not previously reported, thus expanding its current glycomic coverage. However, a targeted LC-MSn analysis was required and best suited to dig even deeper into validating the occurrence of very minor structural isomers carrying the Lewis Y
epitope implicated by positive antibody binding.