Dowex 50w cation exchange resin

The O-linked glycans of WT perlecan DII were released from proteins via slightly modified β-elimination using alkaline borohydride (22 (link)). The sample was desalted by passage through DOWEX 50W cation exchange resin (Sigma). Glycans were additionally purified by porous graphitized carbon (Agilent) solid phase extraction prior to permethylation. Permethylation was carried out in spin columns (Harvard Apparatus) as described (23 (link)). Purification of permethylated oligosaccharides was performed by liquid-liquid extraction with dichloromethane and 0.5 M aqueous sodium chloride. Qualitative analysis was performed via direct infusion via nanoelectrospray (Advion Nanomate, Ithaca, NY) into a Thermo LTQ (San Jose, CA) ion trap mass spectrometer.

Sulfated fucan fraction FS28 [38 (link)] (100 mg) was passed through a Dowex 50-W cation exchange resin (H⁺ 200–400 mesh, Sigma-Aldrich), eluted with water, collected over 20% (v/v) pyridine, dialysed against distilled water (MCWO 6–8 kDa) and then freeze-dried. The pyridine salt was then re-suspended at 1% (w/v) in anhydrous pyridine, and 10% (v/v) total volume of chlorotrimethysilane (CTMS) was added. The solution was incubated at 100°C for 3 h. Samples were taken at different incubation times (DS0, before incubation; DS1 and DS2, at 1 and 2 h into the incubation, respectively). The reaction was ended at sampling time points by dropwise addition of distilled water to destroy excess CTMS. All solutions were then dialysed (MWCO 3.5 kDa) sequentially against running tap water, distilled water, 0.1 M NaCl (to remove any unreactive pyridine salts) and distilled water and freeze-dried. Sulfate concentrations were measured as equivalents of FS28, by colorimetric quantification using Azure A (Sigma). Polysaccharide samples (50 μl, concentrations ranging from 0.3 to 5 mg/ml) were mixed with 50 μl 2.5 M H₂SO₄, before addition of 900 μl of Azure A (20 μg/ml) and determination of absorbance at 620 nm in a microtitre plate reader (200 μl/well). Absorbances were measured against a standard curve of FS28 with concentrations ranging from 0 to 1 mg.