β n acetylglucosaminidase from xanthomonas manihotis

The Ts4 mAb (mouse IgM) and 6035 (a mAb for TEX101 peptide portion (mouse IgG2a)) were established and purified as previously described [2 (link)]. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG polyclonal antibody (pAb) and control mouse IgM were obtained from DAKO (Glostrup, Denmark). HRP-conjugated goat anti-mouse IgM pAb were purchased from Chemicon (Temecula, CA). Various biotin-labeled lectins, such as Dolichos biflorus agglutinin (DBA), Datura stramonium agglutinin (DSA), Phaseolus vulgaris erythroagglutinin (PHA-E), Phaseolus vulgaris leucoagglutinin (PHA-L), Pisum sativum agglutinin (PSA), Sophora japonica agglutinin (SJA), Triticum vulgaris (wheat germ) agglutinin (WGA) were purchased from Vector Laboratories (Burlingame, CA). HRP-conjugated streptavidin was obtained from Invitrogen (Carlsbad, CA). Endoglycosidases (N-glycanase, endoglycosidase-F1, endoglycosidase-F2, endoglycosidase-F3, and endoglycosidase-H) and exoglycosidases (α-mannosidase, β-mannosidase, β-N-acetylglucosaminidase (from Canavalia ensiformis), neuraminidase, and α-L-fucosidase) were from Sigma-Aldrich (St. Louis, MO). β-N-acetylglucosaminidase (from Xanthomonas manihotis) was purchased from New England Biolabs (Ipswich, MA). Other chemicals used in this study were obtained commercially and were of the highest purity available.

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.