Figure 2.

Actions of purified BbhI, BbhIV, and BbhV on O-glycans of porcine gastric mucin (PGM).A, the representative full mass spectra of permethylated nonsulfated O-glycan alditols (m/z 500–3000) obtained from PGM incubated with the purified recombinant BbhI, BbhIV, or BbhV for 24 h. Nontreated PGM was also analyzed for comparison. Pink and light green arrowheads indicate the ion peaks whose estimated amounts decreased and increased, respectively, upon enzymatic treatment (see Table S1 for glycan composition and semiquantification). Asterisks indicate LNFP I added as an external control. B, volcano plots comparing the estimated amount of each O-glycan peak obtained from the BbhI, BbhIV, or BbhV-treated sample with that of the nontreated sample. Data obtained in three independent experiments were used for the analysis. Fold changes and their q-values were used for plotting to identify peaks whose abundances changed by more than 1.5-fold with q-values of < 0.15 post treatment. The q-values are the adjusted p-values obtained by multiple t tests with false discovery rate correction (74). The m/z values of the peaks of interest are shown. C, tandem mass spectra (MS/MS) of the m/z 1025 peak obtained from the nontreated (left panel) and BbhIV-treated (right panel) samples. Predicted O-glycan isomers are shown with their fragmentation patterns. Glycan symbols are depicted according to the nomenclature committee of the Consortium for Functional Glycomics (72).