Protein Protocols Protein Protocols

838 Weitzhandler et al.
2. Add 0.1 U of Jack bean β-N-acetylhexosaminidase in 2 µL of 25 mM sodium citratephosphate
buffer, pH 5.0.
3. Incubate the digest for 20 h at 37°C.
4. Inject 10 µL of each digest directly onto the column.
3.2. PNGase F Digestion
1. Reconstitute approx 100 µg of a monoclonal IgG in 10 µL of 25 mM sodium citratephosphate
buffer, pH 5.0.
2. Add 2 µL of the PNGase F preparation.
3. To an identifical PNGase F digest of the same monoclonal IgG, add 0.1 U of Jack bean
β-N-acetylhexosaminidase in 2 µL of 25 mM sodium citrate-phosphate buffer, pH 5.0.
4. Separately inject 10 µL of each digest directly onto the column.
3.3. Chromatography and Detection of Carbohydrates
Separations of monosaccharides and oligosaccharides can be achieved using a
Dionex BioLC system equipped with a CarboPac MA1 column (4 × 250 mm) and a
CarboPac MA1 guard column working at an isocratic concentration of 480 mM NaOH
and a flow rate of 0.4 mL/min at ambient temperature over 35 min. Separated mono- and
oligosaccharides are detected by PAD with a gold electrode and triple-pulse amperometry
(E 1 = 0.05 V, t 1 = 420 ms; E 2 = 0.80 V, t 2 = 360 ms; E 3 = –0.15 V, t 3 = 540 ms),
measuring at 1000 nA full scale. Alternatively, a more recently described quadruple
potential waveform can be used (Dionex Technical Note 21; Waveform A). (E1 =
+0.1 V, t1 = 400 ms. The first 200 ms is t del and the second 200 ms is t det; E2 =
–2.0 V, t2 = 10 ms; E3 = +0.6 V, t3 = 40 ms; E4 = –0.1 V, t4 = 60 ms).
4. Notes
1. It is essential to use high quality water of high resistivity (18 MeΩ) and to have as little
dissolved carbon dioxide in the water as possible. Biological contamination should be
absent. The use of fresh Pyrex glass-distilled water is recommended. The still should
be fed with high-resistivity (18 MeΩ) water. The use of plastic tubing in the system should
be avoided, as plastic tubing often supports microbial growth. Degas appropriately.
2. It is extremely important to minimize contamination with carbonate. Carbonate, a divalent
anion at pH ≥ 12, binds strongly to the columns and interferes with carbohydrate
chromatography. Thus carbonate is known to affect column selectivity and produce a loss
of resolution and efficiency. Commercially available NaOH pellets are covered with a
thin layer of sodium carbonate and should NOT be used. Fifty percent (w/w) sodium
hydroxide solution is much lower in carbonate and is the preferred source for NaOH.
Diluting 104 mL of a 50% NaOH solution into 2 L of water yields a 1.0 M NaOH solution.
Degas appropriately.
3. This enzyme has a broad specificity, cleaving nonreducing terminal β-N-acetylglucosamine
residues (GlcNAc) and β-N-acetylgalactosamine (GalNAc) with 1–2,3,4, and 6 linkages.
4. PNGase F is an amidase from Flavobacterium meningosepticum. The enzyme cleaves
between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and
complex oligosaccharides from N-linked glycoproteins.
Acknowledgment
We thank Sylvia Morris for her work on the manuscript.