Protein Protocols Protein Protocols

836 Weitzhandler et al.
Fig. 1. HPAEC-PAD of β-N-acetylhexosaminidase digests of oligosaccharide standards. (A)
Digestion of an asialo agalacto biantennary oligosaccharide standard. (B) Digestion of an asialo
agalacto tetraantennary oligosaccharide standard. Peaks 1, 3, and 5 refer to structures 1, 3, and
5 identified in Table 1. (Bottom tracings in A and B: dashed line, chromatography of substrate;
solid line, chromatography of digestion products.)
ture 2; see also Fig. 2B, peak 2). Thus, the two N-linked oligosaccharide peaks
released from the monoclonal IgG are fucosylated and have terminal GlcNAc. To
obtain further structural information regarding GlcNAc linkages, one could use the
Streptococcus pneumoniae β-N-acetylhexosaminidase, an exoglycosidase which
shows much more efficient cleavage of the GlcNAcβ(1→2)Man if the Man residue is
not substituted with GlcNAc at the C-6 (13). This method is also useful for monitoring
the β-N-acetylhexosaminidase preparation for contaminating exoglycosidase
activities as other monosaccharide digestion products would be readily discernible.
2. Materials
1. HPLC grade deionized water (see Note 1).
2. 50% NaOH solution (w/w) (Fisher Scientific, Pittsburgh, PA) (see Note 2).
3. Reference-grade monosaccharides (Pfanstiehl Laboratories, Waukegan, IL).
4. Oligosaccharide standards Man3GlcNAc2 (Table 1, structure 1) fucosylated Man3GlcNAc2 (Table 1, structure 2), asialo agalacto biantennary (Table 1, structure 3), asialo agalacto
biantennary, core fucose (Table 1, structure 4), and asialo agalacto tetraantennary oligosaccharide
(Table 1, structure 5) (Oxford GlycoSciences, Abingdon, UK).
5. β-N-acetylhexosaminidase, Jack bean (Oxford GlycoSciences) (see Note 3).
6. PNGase F (New England BioLabs, Beverly, MA) (see Note 4).
7. 25-mL Plastic pipets (Fisher Scientific).
8. 1.5-mL Polypropylene microcentrifuge tubes, caps, and O rings (Sarstedt, Newton, NC).