Protein Protocols Protein Protocols

Sequencing Heparan Sulfate Saccharides 897
2. Dissolve directly in 10–25 µL of formamide containing freshly prepared 400 mM 2-AA
(54.8 mg/mL) and 200 mM reductant (sodium cyanoborohydride; 12.6 mg/mL) and incubate
at 37°C for 16–24 h in a heating block or oven. (Caution: The reductant is toxic
and should be handled with care.) The volume used should be sufficient to provide a
500–1000-fold molar excess of 2-AA over saccharide (see Note 1).
3. Remove free 2-AA, reductant, and formamide from the labeled saccharides by gel filtration
chromatography (Sephadex G-25 Superfine). Dilute the sample (maximum 250 µL of
reaction mixture) to a total of 1 mL with distilled water (see Note 2).
4. Load sample onto two 5-mL HiTrap Desalting columns (Pharmacia Ltd.) connected in
series. Alternatively it is possible to use self-packed columns of other dimensions.
5. Elute with distilled water at a flow rate of 1 mL/min and collect fractions of 0.5 mL.
Saccharides consisting of four or more monosaccharide units typically elute in the void
volume (approx fractions 7–12). Note that the HiTrap columns can be eluted by hand
with a syringe without need for a pump.
6. Pool and concentrate these fractions by centrifugal evaporation or freeze drying.
3.1.2. Labeling Saccharides with ANDSA
1. Dry down the purified saccharide (typically 2–10 nmol) in a microcentrifuge tube by centrifugal
evaporation.
2. Dissolve directly in 10 µL of formamide.
3. Mix with 15 µL of formamide containing ANDSA at a concentration of 80 mg/mL
(approaching saturation) and incubate at 25°C for 16 h.
4. Mix with 10 µL of formamide containing 1 mg of the reductant sodium triacetoxyborohydride
(see Note 1) and incubate for 2 h at 25°C.
5. Remove free ANDSA by gel filtration as described in Subheading 3.1.1. for 2-AA.
3.2. Nitrous Acid Treatment of Saccharides
Low pH nitrous acid cleaves HS only at linkages between N-sulfated glucosamine and
adjacent hexuronic acid residues (17,18). Under mild controlled conditions nitrous acid cleavage
creates a ladder of bands corresponding to the positions of internal N-sulfated glucosamine
residues in the original intact saccharide (11). A series of different reaction stop points are
pooled, resulting in a partial digest with a range of different fragment sizes.
1. Dry down 1–2 nmol of labeled saccharide by centrifugal evaporation.
2. Redissolve in 80 µL of distilled water and chill on ice.
3. Add 10 µL of 200 mM HCl and 10 µL of 20 mM sodium nitrite (both prechilled on ice)
and incubate on ice.
4. At a series of individual time points (typically 15, 30, 60, 120, and 180 min), remove an
aliquot and stop the reaction by raising the pH to approx 5.0 by the addition of 1/5 volume
of 200 mM sodium acetate buffer, pH 6.0 (see Note 3).
5. Pool the set of aliquots and either use directly for enzyme digests or desalt as described in
Subheading 3.1.
3.3. Exoenzyme Treatment of Saccharides
The approach for treatment of HS samples with exoenzymes is described below.
Details of the specificities of the exoenzymes is given in Table 1. These enzymes have
differing optimal pH and buffer conditions, but in general they can be used under the
single set of conditions given here, which simplifies the multiple enzyme treatments
required (see Note 4).