2.3 High-pH reversed-phase separation
UPLC loading buffer was used to redissolve the digested peptides, an ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 mm × 150 mm, Waters, USA) was used to separate them at a flow rate of 200 μL·min–1. The mobile phase contained 2% acetonitrile (A, pH=10) and 80% acetonitrile (B, pH=10) with a gradient elution: 0–2 min, 0–5% solvent B; 2–17 min, 5% solvent B; 17–18 min, 5–10% solvent B; 18–35.5 min, 10–30% solvent B; 35.5–28 min, 30–36% solvent B; 38–39 min, 36–42% solvent B; 39–40 min, 42–100% solvent B. Finally, 14 fractions were created by combing a total of 28 fractions were collected according to their peak type and time.