Increasing the depth of mass spectrometry-based glycomic coverage by additional dimensions of sulfoglycomics and target analysis of permethylated glycans

Abstract

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.

Efficient Mapping of Sulfated Glycotopes by Negative Ion Mode nanoLC–MS/MS-Based Sulfoglycomic Analysis of Permethylated Glycans

“…The former set of ions resulting from concerted cleavages at the GalNAcitol (see schematic drawing in Fig. 2) has been previously observed under negative ion mode MALDI-MS/MS of sulfated O-glycans 21,23 , which firmly establishes the 6-arm substituents. It is clear that an isomeric structure II carrying instead a non-fucosylated sulfated LacNAc on the 6-arm also existed, as indicated by the B ions at m/z 528, its analogous satellite ions at m/z 588, 616, 632, 676, 745, and 759, and the Z 1 and Y 1 ions at m/z 789 and 807.…”

Carbohydrate Sequence of the Prostate Cancer-associated Antigen F77 Assigned by a Mucin O-Glycome Designer Array

“…In separate experiments, 4M-f1 was shown to be bound by a monoclonal anti-H type 2 but not anti-H type 1 (data not shown). Taking into account the F77 antigen activity of the branched blood group B glycolipid BIII and the lack of detection of type 1 backbones in a detailed study of the O -glycans of PSM (28), the proposed oligosaccharide sequence of 4M-f1 is a blood group H on a branched backbone of the I-antigen type (29), originating from the C6 position of the core GalNAcol as depicted in Scheme 1. Thus, we propose that the parent O -glycan alditol before periodate oxidation was at least the octasaccharide shown in Scheme 2.…”

O-Glycome Beam Search Arrays for Carbohydrate Ligand Discovery

“…The microarray data in the small-scale experiments, using as little as ϳ1 mg of O-glycan alditols, showed that ligand-containing fractions can be readily detected by this approach with associated MALDI-MS monitoring. The ligand-positive hexasaccharide with the blood group H type 1 sequence (rather than H type 2) has not been described in PSM to our knowledge (37). With optimization of the O-glycan derivatization step there is scope for a substantial increase of yields.…”