O
-Linked Chain Release and Fractionation 181
181
15
O
-Linked Oligosaccharide Chain Release
and Fractionation
Elizabeth F. Hounsell
1. Introduction
O-linked chains of glycoproteins have classically been released by alkaline boro-
hydride degradation, in which mild alkali (0.05 M OH
–
) is used to cause β-elimination
from the β carbon of serine (R-H) or threonine (R-CH
3
) in the protein backbone.
To inhibit subsequent elimination around the glycosidic ring of the linkage monosac-
charide, and on backward down the oligosaccharide if linked at C-3, the reaction is
carried out in the presence of 1 M sodium borohydride to give simultaneous reduction
of the reducing sugar formed after elimination. An advantage of this technique is that
there is now a large database of nuclear magnetic resonance (NMR) chemical shifts
for mucin-type oligosaccharide alditols (1,2) that can be searched in a computer-as-
sisted way (3) for structural identification. The disadvantage of this technique is that
the resulting alditol is not capable of undergoing a reductive amination procedure for
coupling to a sensitive flourescent label or for polyvalent coupling to lipid or protein
for subsequent immunoassay. However, it is possible to reoxidize selectively the
alditol using periodate, which results in a new aldehyde being formed for reductive
amination, and, indeed, this is the basis for a method for structural analysis by thin-
layer chromatography-mass spectometry (MS) (4). Note, however, that for subsequent
biological or immunological assay, any branching at GalNAc-Ser/Thr, often found in
mucins, is destroyed by this procedure.

From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
182 Hounsell
If branching is not present, a useful cleavage can be obtained by the enzyme O-
glycanase (α-N-acetylgalactosaminidase). This enzyme has the disadvantage that only
relatively simple oligosaccharides are released with any reproducibility (5,6); the dis-
accharide Galβ1-3GalNAcα1-Ser/Thr is the best substrate. Hydrazinolysis is prob-
ably the preferred technique for release of all oligosaccharides in both the presence
and the absence of sulfate and/or sialic acids, if the conditions are optimized. It may be
a good idea to carry out this reaction in triplicate with the alkaline borohydride degra-
dation and O-glycanase in order to ensure that the complete picture of a mucin at a
structural level is obtained. Note that if the protein is required for analysis, rather than
the oligosaccharide, mild alkali in the absence of borohydride (7) or hydrolysis with
trifluoromethanesulfonic acid (TFMSA) can be used to keep the protein intact.
After the release of oligosaccharide alditols, these can be fractionated by normal- or re-
versed-phase high-performance liquid chromatography (HPLC) (8) or anion-exchange chro-
matography, e.g., high-performance anion-exchange chromatography (HPAEC) on two
CarboPac PA-100 columns (Dionex Camberley, Surrey, UK) in series (9). Sulfated and
sialylated oligosaccharide alditols can also be separated by these techniques; however, for
better results the reversed-phase HPLC should be by porous graphitized carbon (PGC) in
0.1% trifluoroacetic acid (TFA) (10,11), and the normal-phase HPLC should be in the pres-
ence of buffers (9). Sulfated oligosaccharide alditols are retained well on the HPLC columns
(12). For reducing oligosaccharides, PGC is again a good alternative for both neutral and
anionic oligosaccharides, which have similar retention times to each other and to their alditols.
On HPAEC (13), the reducing oligosaccharides will be retained significantly longer than the
alditols, to give improved chromatography. Sulfated oligosaccharides may now be retained
too long on a CarboPac PA-100 column. Although HPAEC with pulsed amperometric detec-
tion (PAD) is a sensitive technique, additional sensitivity can be obtained by flourescent
labeling. The usual labels, 2-aminopyridine (14,15) and 2-aminobenzamide (15,16) cause

sulfated oligosaccharides to be retained too long on reversed phase or PGC column. For
neutral or sialylated oligosaccharides, separation is primarily achieved by reversed-phase
and normal-phase chromatography, often as a two-dimentional map (14,15), or on weak ion
exchange, e.g., GlycoSep C™ (Oxford GlycoSciences, Abingdon, Oxford, UK) (17). Neu-
tral 2-aminobenzamide (2-AB) oligosaccharides (naturally occuring or after desialylation)
can be analyzed by gel filtration in water as eluent (usually Bio-Gel P4 chromatography) to
give molecular size estimation. This is particularly useful to follow exoglycosidase diges-
tions to obtain additional sequence information. More information about these techniques
can be obtained from refs. 18–20. The use of electrospray ionization MS coupled with colli-
sion-induced dissociation MS is another possibility for analysis at the glycopeptide level
(21). The internet provides a source for predicting potential O-glycosylation sites on proteins
at />2. Materials
2.1. Alkaline Borohydride Degradation (
β
-Elimination)
and Chromatography
1. 1 M NaBH
4
in 0.05 M NaOH made up fresh.
2. Glacial acetic acid.
O
-Linked Chain Release and Fractionation 183
3. Methanol.
4. Cation-exchange column Dowex 50W X8 H
+
form.
5. Phenyl boronic acid (PBA) Bond Elut columns (Jones Chromatography, Hengoed, UK)
activated with MeOH (22).
6. 0.2 M NH
4

OH.
7. 0.01, 0.1, and 0.5 M HCl.
8. HPLC apparatus fitted with an ultraviolet detector (approx 1 nmol of mono- and oligosac-
charides containing N-acetyl groups can be detected at 195–210 nm) and pulsed electro-
chemical detector (oligo- and monosaccharides ionized at high pH can be detected at
picomole level).
9. Columns: reversed-phase C
18
, amino bonded silica, PGC (Hypercarb 7µ, Hypersil Ltd.,
Runcorn Cheshire, UK), CarboPac PA100 and CarboPac PA1 (Dionex Camberley).
10. Eluents for HPLC: HPLC grade water, acetonitrile, 0.1% aqueous TFA; acetonitrile con-
taining 0.1% TFA, ammonium formate.
11. Eluents for HPAEC: 12.5 M NaOH (BDH, Poole, Dorset, UK) diluted fresh each day to
200, 100, 80, and 1.5 mM. After chromatography and detection, salt needs to be removed
by a Dionex micromembrane suppressor or by cation-exchange chromatography before
further analysis, e.g., by methylation.
12. Eluent A: 0.08 M NaOH.
13. Eluent B: 0.5 M sodium acetate (Aldrich, Poole, Dorset, UK) in 0.08 M NaOH.
2.2. Reoxidation
1. Sodium periodate, analytical reagent grade.
2. Imidazole (Sigma, Poole, Dorset, UK) 40 mM adjusted to pH6.5 with HCl
3. Butan-2,3-diol.
2.3. Release of the Core 1 Disaccharide (Gal
β
1-3GalNAc
α
–) from Mucins
1. Bio-Spin
®
chromatography columns (Bio-Rad, Hercules CA), or home made columns

suitable for 1.5-mL microcentrifuge tubes packed with 0.8 mL of Bio-Gel P30 acrylamide
gel matrix . Store at 4°C in 0.15 M sodium chloride-17.5 mM sodium citrate, pH 7.0,
containing 0.2% w/v sodium azide as preservative.
2. O-Glycanase
®
; Streptococcus (Diplococcus) pneumoniae Endo-α-N-acetylgalacto-
saminidase (EC 3.2.1.907) (Oxford GlycoSciences). Store at –20°C for up to 6 mo, but
avoid repeated freeze-thawing.
3. 0.1 M sodium citrate-phosphate, pH 6.0; make up with HPLC-grade water.
4. Dowex 50X-12 H
+
-form resin.
5. Sephadex GM25.
2.4. Hydrazinolysis
1. 0.5-mL screw-capped V-bottomed Reacti-vials™ (Pierce and Warriner, Chester, UK).
2. Anhydrous hydrazine (Pierce and Warriner).
3. Whatman CF-11 cellulose chromatography medium.
4. Reagent A: butanol:ethanol:acetic acid, 4:1:0.5 (v/v/v).
5. Reagent B: butanol:ethanol:water, 4:1:1 (v/v/v).
6. Reagent C: acetic anhydride:methanol, 2:5 (v/v).
7. Reagent D: 0.2 M sodium acetate.
8. Sep-Pak C
18
cartridge (Waters, Watford, UK).
9. Alternatively, the Glycorelease N- and O-Glycan recovery kit (Oxford GlycoSciences)
can be used.
184 Hounsell
2.5. Isolation of the Protein by TFMSA Destruction of Oligosaccharides
1. Anhydrous TFMSA (23).
2. Reagents as specified in Glyco Free™ Deglycosylation kit (K500, Oxford, Glyco-

Sciences).
2.6. 2-AB Fluorescence Labeling and Size Exclusion
Chromatography (SEC)
1. Signal labeling kit (K404,Oxford GlycoSciences).
2. Dowex AG50 X 12 (H
+
form).
3. Dowex AG1 X 8 (acetate form).
4. RAAM 2000 Glycosequencer (Oxford GlycoSciences) or Biogel P4 column (100 × 2 cm)
in a water jacket at 55°C and HPLC pump with refractive index and fluorescence detectors.
5. GlycoSepH™ and GlycoSepC™ HPLC column (Oxford GlycoSciences).
3. Methods
3.1. Alkaline Borohydride Degradation (
β
-Elimination)
1. Release O-linked chains by treatment with 0.05 M sodium hydroxide in the presence of 1 M
NaBH
4
or NaB[
3
H]
4
for 16 h at 50°C.
2. Degrade excess NaBH
4
or NaB[
3
H]
4
by the careful addition with the sample on ice of

glacial acetic acid (to pH 7.0) or acetone (1 mL/100 mg of NaBH
4
) followed by repeated
evaporation with methanol.
3. Desalt on a cation-exchange column and analyze by reversed-phase HPLC (Subheading
3.6.2.) or HPAEC (Subheading 3.7.1.).
4. Or, for microscale identification of the presence of alditols, dissolve the sample in 200 µL
0.2 M NH
4
OH and add to the top of a PBA minicolumn prewashed with MeOH, water,
and 0.2 M NH
4
OH.
5. Wash the PBA column with 2 × 100 µL 0.2 M of NH
4
OH and 2 × 100 µL of water.
6. Specifically elute the alditols in 1 M acetic acid.
7. Evaporate the sample and reevaporate with 2 × 100 µL of water.
3.2. Oxidation of Oligosaccharide Alditols
1. To the dry alditols, add twice the molar ratio of sodium periodate in imidazole buffer (see
Note 1).
2. Oxidize in the dark at 0°C for 5 min.
3. Destroy excess oxidant with butan-2,3-diol (two times the molar excess over periodate)
for 40 min at 0°C in the dark.
3.3. Release of Core 1 Disaccharide (Gal
β
1-3GalNAc
α
–) from Mucin
Using Endo-

α
-
N
-Acetylgalactosaminidase (
O
-Glycanase)
3.3.1. The Removal of Glycerol from
O
-Glycanase (
see
Note 2)
1. Invert a Bio-Spin P30 polyacrylamide BioGel column (0.8 mL column volume) several
times and allow the buffer to drain by gravity.
2. Wash the column with 300 µL of 0.01 M citrate-phosphate, pH 6.0, place in a collection
tube, and centrifuge for 2 min at 1100g. Repeat four times.
3. Make up 6 mU O-Glycanase to 100 µL with 0.01 M citrate-phosphate, pH 6.0, and load
the sample carefully and directly to the center of the column, drop wise (see Note 3).
4. Centrifuge for 4 min at 1100g and collect the excluded O-Glycanase.
O
-Linked Chain Release and Fractionation 185
5. Pass the excluded O-Glycanase through a second Bio-Spin column to maximize glycerol
removal.
3.3.2. Hydrolysis of Gal
β
1-3GalNAc
α
- from mucin using
O
-Glycanase
1. Reconstitute mucin (100 µg) with 90 µL of 0.1 M citrate-phosphate, pH 6.0, containing

100 µg/mL of bovine serum albumin and 0.02% (w/v) sodium azide. Mix well.
2. Add 0.6 mU of deglycerolyated O-Glycanase (10 µL).
3. Incubate for 18 h at 37°C (see Note 4).
4. Load reactions on Dowex 50X-12H
+
form resin (3 × 0.5 cm column) and elute with three
column vol of HPLC-grade water.
5. Collect the effluent and eluent and then pool (2.5-mL volume).
6. Load onto a PD10-Sephadex GM25 column and elute with HPLC-grade water to isolate
liberated disaccharide from intact mucin.
3.4.
O
-Linked Oligosaccharide Release by Hydrazinolysis
1. Dry salt-free glycoprotein into a V-bottomed Reacti-vial and remove from the lyophilizer
immediately before the reaction is due to commence.
2. Using a clean, dry, acid-washed glass pipet, transfer 100 µL of anhydrous hydrazine to
the vial and cap immediately. Incubate at 60°C for 5 h (see Note 5).
3. Allow the Reacti-vial to cool to room temperature, and transfer the reaction mixture to a
1-mL cellulose (Whatman CF-11) microcolumn washed with reagent A.
4. Wash the column with 3 × 1mL reagent B.
5. Re-N-acetylate the glycans on the column by adding 1.4 mL of reagent C for 30 min at
room temperature.
6. Wash the column with 4 × 1 mL of reagent B followed by 1 mL of methanol (see Note 6).
7. Elute the oligosaccharides with 2 × 1 mL of reagent D.
8. Complete the re-N-acetylation with 0.1 mL of acetic anhydride for 30 min at room tem-
perature.
9. Wash a Sep-Pak C
18
cartridge with 2 mL of methanol and 2 mL of H
2

O.
10. Transfer the sample containing O-glycans to the cartridge and collect the eluate. Elute the
remaining glycans with 0.5 mL of H
2
O.
3.5. TFMSA Treatment
1 Make up a 3 g/mL solution of TFMSA in anisole and cool in dry ice/ethanol
2. Add 10 times the weight of TFMSA in anisole to the lyophilized material in a teflon
screw-capped vial standing on a bed of ice.
3. Incubate at 0°C for 6–16 h with occasional vigorous shaking.
4. With the vial on ice, add 1 vol of cold anhydrous diethyl ether and then add this mixture
to 1 vol of a frozen slush of aqueous pyridine.
5. Warm the solution to room temperature and extract with ether.
6. Collect the aqueous phase containing the peptide with partial glycosylation depending on
the reaction time.
7. Alternatively, follow the instructions in the GlycoFree kit.
3.6. HPLC of 2-AB-Labeled Oligosaccharides
3.6.1. Preparative HPLC on a GlycoSep C HPLC Column
1. Wash the column with water for 30 min at a flow rate of 0.4 mL/min.
2. Wash the column with acetonitrile for 30 min at a flow rate of 0.4 mL/min.