Propac pa 1

High performance anion exchange chromatography (HPAEC) was performed on each of the pooled labeled disaccharides as follows: 1 mL of the pooled samples in 150 mM NaOH (aq.) was injected onto a ProPac PA-1 analytical column (4 × 250 mm, ThermoFisher Scientific, Loughborough, UK) pre-equilibrated in 150 mM NaOH (aq.). The column was held under isocratic flow (150 mM NaOH (aq.)) for 11 min prior to developing a linear gradient from 0 to 1 M NaCl (in isocratic 150 mM NaOH (aq.)) over 30 min. Standards, comprising the BODIPY-labeled 8 common disaccharides found in heparin and heparan sulphate (Figure 3), were also run under identical conditions prior to the samples and afterwards.

A strong anion-exchange column Pro Pac PA1 (9 × 250 mm, Thermo Fisher Scientific) was used to determine the purity of ¹³C-labeled disaccharides after purification. Mobile phase A was 3 mM NaH₂PO₄, pH 3.0. Mobile phase B was 3 mM NaH₂PO₄ and 1 M NaCl, pH 3.0. The gradient was as follows: 0–20 min 5–20% B, 20–72 min 20–95% B, and 72–75 min 95–100% B with a flow rate of 1 mL min⁻¹. The ultraviolet absorbance at 232 nm was scanned and recorded. The different retention times of two groups of isomers (Δ[¹³C]UA2S-GlcNS and Δ[¹³C]UA-GlcNS6S; Δ[¹³C]UA2S-GlcNAc and Δ[¹³C]UA-GlcNAc6S) on SAX-HPLC were determined by comparing with the retention times of the native disaccharide standards (Iduron) on SAX column.
ESI–MS (Thermo Scientific TSQ Fortis) analysis was used to confirm the MW of each ¹³C-labeled disaccharide. The ESI–MS analysis was performed in the negative-ion mode and with the following parameters: Neg ion spray voltage at 3.0 kV, sheath gas at 15 Arb, ion transfer tube temp at 320 °C, and vaporizer temp at 100 °C. The mass range was set at 240–800.

Heparan sulfate disaccharides of HSPGs were analyzed as described previously (Carnachan and Hinkley, 2017 (link)). Briefly, proteins (1 mg/ml) were digested in 200 µl digestion buffer (100 mM NaOAc, 2 mM CaOAc, pH 7.0) with 1.75 mIU of Heparinase I + II and III (NEB) for 16 hr at 30°C. Heparinases were inactivated at 95°C for 10 min and denatured proteins were pelleted at 16,000× g for 10 min; 100 µl of supernatant was analyzed by HPLC with a strong anion exchange column (ProPac PA1, Thermo Fisher) with an elution gradient of 2 M NaCl, pH 3.5. Disaccharides were detected at an absorbance maximum of 232 nm. A standard mixture of heparan sulfate disaccharides (20 µg/ml; Iduron, UK) was analyzed to identify heparan sulfate disaccharides.