β1 4 galactosidase from bacteroides fragilis

The 2-AA labeled glycans were sequentially digested using the following exoglycosidases according to the manufacturers’ instructions: α2-3,6,8 neuraminidase from Clostridium perfringens (New England Biolabs, Hertfordshire, UK), α2-3 neuraminidase from Streptococcus pneumoniae (New England BioLabs, Hertfordshire, UK), β1,4-galactosidase from Bacteroides fragilis (New England Biolabs, Hertfordshire, UK), and α-L-fucosidase from bovine kidney (Sigma-Aldrich, Dorset, UK), β-N-acetylglucosaminidase from Xanthomonas manihotis (New England Biolabs, Hertfordshire, UK), and α(1–2,3,6)-mannosidase from Jack bean (Sigma-Aldrich, Dorset, UK). Endoglycosidase H (endoH) from Streptomyces picatus (New England BioLabs, Hertfordshire, UK) was used for quantification of oligomannose structures. Prior to chromatographic analysis, polyvinylidene difluoride (PVDF) protein-binding membrane plates (Millipore, Feltham, UK) were used for the removal of enzymes.

The 2-AA–labeled glycans were sequentially digested using the following exoglycosidases: α2–3,6,8 neuraminidase from Clostridium perfringens (New England Biolabs), β1,4-galactosidase from Bacteroides fragilis (New England Biolabs), α-l-fucosidase from bovine kidney (Sigma-Aldrich), β-N-acetylglucosaminidase from Xanthomonas manihotis (New England Biolabs), and α(1–2,3,6)-mannosidase from jack bean (Sigma-Aldrich). Endoglycosidase H from Streptomyces picatus (New England Biolabs) was used for quantification of oligomannose structures. Digestions were carried out in an incubation buffer (50 mm sodium phosphate, pH 5.0) at 37 °C for 16 h. PVDF protein-binding membrane plates (Millipore) were used for removal of enzymes prior to HILIC-UPLC analysis.