Chromeleon 7.2 cds

5 mg of AMP and 1 mL of trifluoroacetic acid (TFA, 2 M) were hydrolyzed at 121°C for 2 h. The mixture was dried with nitrogen, and then washed with methanol 2–3 times followed. The monosaccharide standards included fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid and glucuronic acid. Finally, samples were analyzed via high-performance anion-exchange chromatography (HPAEC) (ICS5000, Thermo Fisher Scientific, United States) with Dionex™ CarboPac™ PA-20 column (150 mm × 3.0 mm, 10 μm). Mobile phase A was 0.1 M NaOH, mobile phase B was 0.1 M NaOH, 0.2 M NaAc. The composition of eluent A was adjusted to 95% at 0 min, 80% at 30 min, 60% at 30.1 min, 60% at 45 min, 95% at 45.1 min, 95% at 60 min. The column temperature was 30°C. The flow rate was 0.5 mL⋅min^–1 and the injection volume was 5 μL. The determination of monosaccharide composition was made with an electrochemical detector and the peaks were processed using Chromeleon 7.2 CDS (Thermo Scientific).

Approximately 5 mg of the sample was hydrolyzed with trifluoroacetic acid (2 M) at 121 °C for 2 h in a sealed tube. Dry the sample with nitrogen. Add methanol to wash, then blow dry, repeat methanol wash 2–3 times. The residue was re-dissolved in deionized water and filtered through 0.22 μm microporous filtering film for measurement. High-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-20 anion-exchange column (3 by 150 mm; Dionex) with a pulsed amperometric detector (PAD; Dionex ICS 5000+ system) was used to analyzed sample. Thirteen standard monosaccharides, namely fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), mannose (Man), glucose (Glc), xylose (Xyl), mannuronic acid (Man-UA), fructose (Fru), ribose (Rib), guluronic acid (Gul-UA), glucuronic acid (Glc-UA), and galacturonic acid (Gal-UA) were used as the references. Data were acquired on the ICS5000+ (Thermo Scientific), and processed using chromeleon 7.2 CDS (Thermo Scientific).

PTFP with 5 mg was weighed accurately and placed in a sealed tube. Trifluoroacetic acid solution was added to the sample for acid hydrolysis, after hydrolysis, nitrogen gas was used to dry the residual PTFP in the tube. Wash with methanol, then blow dry and repeat methanol washing 2–3 times. The residue was dissolved in deionized water and passed through 0.22 μm microporous membrane filtration. The PTFP filtrate was analyzed by high-performance anion exchange chromatography on a CarboPac PA-20 anion exchange column using a pulse current detector. Data were collected on ICS5000+ (Thermo Technology) and processed using chromeleon 7.2 CDS (Thermo Science).

The monosaccharide composition of GSP-1a was determined via high-performance anion-exchange chromatography (HPAEC). Briefly, 5 mg of the polysaccharide sample was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 121 °C for 2 h in a sealed tube. The polysaccharide sample was dried with nitrogen then supplemented with methanol and blown dry to remove TFA. This process was repeated three times. The residue was re-dissolved in deionized water and filtered through 0.22 μm microporous filtering film for measurement. The processed sample was analyzed via high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-20 anion-exchange column (3 × 150 mm; Dionex, Sunnyvale, CA, USA) using a pulsed amperometric detector (Dionex ICS 5000 system, Thermo Fisher Scientific, Waltham, MA, USA) by Sanshu Biotech. Co., Ltd. (Shanghai, China). Flow rate, 0.5 mL/min; injection volume, 5 μL; solvent system A: (ddH₂O), solvent system B: (0.1 M NaOH); solvent system C: (0.1 M NaOH, 0.2 M NaAc). The gradient program volume ratio of solution A, B, and C was 95:5:0 at 0 min, 85:5:10 at 26 min, 85:5:10 at 42 min, 60:0:40 at 42.1 min, 60:40:0 at 52 min, 95:5:0 at 52.1 min, and 95:5:0 at 60 min. Data were acquired on the ICS5000 (Thermo Fisher Scientific, Waltham, MA, USA) and processed using Chromeleon 7.2 CDS (Thermo Scientific, Waltham, MA, USA).

5 mg of sample was hydrolyzed with trifluoroacetic acid (TFA, 2 M) at 121°C for 2 h in a sealed tube. The sample was dried with nitrogen. Add methanol to wash, then blow dry, repeat methanol wash 2–3 times. The monosaccharide standards included fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid and glucuronic acid. Finally, samples were analyzed by high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-20 anion-exchange column (3 by 150 mm; Dionex) using a pulsed amperometric detector (PAD; Dionex ICS 5000 system). Flow rate, 0.5 mL/min; injection volume, 5 μL; solvent system A: (ddH2O), solvent system B: (0.1 M NaOH), solvent system C: (0.1 M NaOH, 0.2 M NaAc); gradient program, volume ratio of solution A, B, C was 95:5:0 at 0 min, 85:5:10 at 26 min, 85:5:10 at 42 min, 60:0:40 at 42.1 min, 60:40:0 at 52 min, 95:5:0 at 52.1 min, 95:5:0 at 60 min. Data were acquired on the ICS5000 (Thermo Fisher Scientific), and processed using chromeleon 7.2 CDS (Thermo Fisher Scientific).

The monosaccharide composition of SIP was analyzed using high-performance anion-exchange chromatography (HPAEC). In brief, 1 mL of 2 M TFA was added to 5 mg of polysaccharide sample, and the mixture was incubated in a 121°C oil bath for 2 h. The sample was soaked in methanol and blown dry with nitrogen to remove residual TFA, and then freeze-dried. The residue was re-dissolved in deionized water and filtered through a .22-μm microporous membrane filter for HPAEC analysis. The sample extracts were analyzed by high-performance anion-exchange chromatography (HPAEC) using a CarboPac PA-20 anion-exchange column (3 by 150 mm; Dionex) connected to a pulsed amperometric detector (PAD; Dionex ICS 5000 system). The column was run at a flow rate of .5 mL/min. Data were acquired by an ion chromatography system (ICS5000, Thermo Scientific) and processed using chromeleon 7.2 CDS (Thermo Scientific).

The samples were analyzed for their monosaccharide composition following the reported method [31 (link)]. Approximately 5 mg of the sample was hydrolyzed in the chromatography vial with 2 M trifluoroacetic acid (TFA) at 121 °C for 2 h and dried in nitrogen. Methanol was added and washed 3 times to dry. The residues were redissolved in deionized water and filtered through a filtering membrane (0.22 µm). Next, the samples were collected for determination by high performance anion exchange chromatography (HPAEC) using Dionex™ CarboPac™ PA20 liquid chromatographic column (150 × 3.0 mm, 10 µm). The HPAEC conditions are described in Supplementary Methods S1. Data were acquired on the ICS5000 (Thermo Fisher Scientific, Waltham, MA, USA) and processed using chromeleon 7.2 CDS (Thermo Fisher Scientific, Waltham, MA, USA).