Gemini nx column

The Shimadzu LC‐20AB HPLC Pump System was used for high‐pH reversed‐phase chromatography. The iTRAQ‐labeled peptide mixtures were reconstituted in 0.5 mL Buffer A (20 mm ammonium formate and 2% ACN, pH 10) and loaded onto a Gemini‐NX column (5 μm, C18, 110 Å, 250 × 4.6 mm; Phenomenex, Guangzhou, China). The peptides were eluted with Buffer A for 10 min and then with 5–30% Buffer B (20 mm ammonium formate and 98% ACN, pH 10) for 15 min. The column was then treated with 30–80% Buffer B for 3 min. All flow rates were 1 mL·min⁻¹.

After adding 6 ml of 1:1 hydrochloric acid to the 0.05 g sample, continue to add 3–4 drops of phenol, freeze for 3–5 min, repeat vacuum‐filling with nitrogen 3 times, and hydrolyze for 22 hr. Open the hydrolysis tube, filter the acid hydrolyzate into a 50‐ml beaker, evaporate it in a water bath, and dilute to 2.0 ml with 20 mmol/L hydrochloric acid. Take 0.5 ml of the hydrolyzed solution into a 10‐ml volumetric flask, add 1 ml of 0.5 M Na₂CO₃ solution, and add 1 ml of 0.1% 2,4‐dinitrofluorobenzene 60°C water bath to derive for 1 hr, and use 0.1 M KH₂PO₄ to make the volume to 10 ml. To be tested after filtration with 0.45 μm inorganic membrane. The amino acid analysis was performed on a Waters 1525 Series HPLC system (Waters Corp., USA) equipped with a binary gradient pump and a VWD detector. The separation was carried out at 37°C using a Phenomenex Gemini‐NX column (250 × 4.6 mm, 5 μm). Mobile phase A was acetonitrile/H₂O (50:50, v/v), and mobile phase B was 0.05 mol/L sodium acetate solution (6.80 g sodium acetate trihydrate was dissolved in 900 ml water, adjust the pH to 4.0–5.0 with glacial acetic acid, and dilute to 1,000 ml with water), gradient elution for 45 min. The flow rate of 1 ml/min, the injection volume was 10 μl, and the detection wavelength was 360 nm.