The MS2 spectral intensity correlation analysis of the [M - H]- ions at m/z 733 that was degraded suggests a core 2 structure with Fucα1-2Galβ1-3(GlcNAcβ1-6)GalNAc configuration because it gives similar spectra to the spectra reported in the MS2 database UniCarb-DB (Table 1). The drop in intensity

of the [M - H]- ions at m/z 733 after hexosaminidase is due to the degradation of the see more terminal HexNAc (Figure 3a) generating a core 1 structure terminating in a blood group H epitope (Fucα1-2Galβ1-3GalNAcol) (Figure 3a), which is also supported with spectrum reported in the MS2 database UniCarb-DB (Table 1). Hence, this drop in intensity in core 2 sequence Fucα1-2-Galβ1-3(GlcNAcβ1-6)GalNAcα1-Ser/Thr generating Inhibitors,research,lifescience,medical core 1 sequence confirmed the terminal HexNAc to be β1-6 linked GlcNAc in the structure. However, the MS2 spectral

correlation Inhibitors,research,lifescience,medical analysis of the [M - H]- ions at m/z 790 with spectra reported in the MS2 database UniCarb-DB suggests that this was a core 2 structure with HexNAc-Galβ1-3(GlcNAcβ1-6)GalNAc configuration (Table 1) with unknown Inhibitors,research,lifescience,medical information about the nature of the HexNAc residue on the C-3 antenna. After hexosaminidase treatment only the C-6 GlcNAc could be removed (Figure 3b). This generated a core 1 structure with one terminal HexNAc still remaining ([M - H]- ions of m/z 587) indicating that the second terminal HexNAc was not in a Inhibitors,research,lifescience,medical β-configuration (Figure 3b), and treatment with the a-N-actetylgalatosaminidase was not successful (data not shown). The MS2 spectral correlation analysis of the [M - H]- ions at m/z 587 suggests a core 1 structure terminated with HexNAc (Table 1) but did not give conclusive result about the configuration (Table 1) when compared with spectra reported in the MS2 database UniCarb-DB. Due to lack of specific enzymes, MS2 of the substrate ([M - H]- ions at m/z 790) and product ([M - Inhibitors,research,lifescience,medical H]- ions at m/z 587) were interpreted manually to investigate the configuration of terminal HexNAc (Figure

3b). Figure 3 (a) Negative ion baseline chromatograms of β-N-acetylhexosaminidase untreated (front) and treated (back) porcine gastric mucin (PGM) oligosaccharides showing the increase of the ions m/z 530 and 587 and a decrease of the m/z 790 and 733 after these … The identification of cross ring 0,2A fragments of the core 1 GlcNAc residue in the MS2 spectra of the substrate at m/z 790 and the product at m/z 587 (Figure 3b) suggests that this was a terminal HexNAc linked to the 4 position of a Gal because extension to the C-4 provides a diagnostic ion of m/z 304 after loss of water, whereas extension of C-3 does not give this fragment [8]. This indicates that the structure of the substrate ([M - H]- ions at m/z 790) and product ([M - H]- ions at m/z 587) is HexNAc1-4Galβ1-3(GlcNAcβ1-6)GalNAcol and HexNAc1-4Galβ1-3GalNAcol respectively.