Rhcd10

OGP was synthesized as described above and resolved in water (1 × 10⁻³ m). OGP (4 µL) was incubated with or without 0.5 µL of rhCD10 (Cat# ab157051, Abcam) in a final volume of 200 µL of 50 × 10⁻³ m Tris‐HCl buffer (pH 7.5) for 1 h at 37 °C. To inhibit the enzymatic activity of rhCD10, rhCD10 was pretreated with 1 × 10⁻⁶ m thiorphan (Cat# ab141169, Abcam) for 30 min before incubation with OGP. The hydrolysis reaction was stopped by heating at 100 °C for 5 min. The peptide products were identified by reverse phase‐HPLC on a Phenomenex‐Pack Gemini‐NX 3 μ C18 110 Å (2.00 mm × 150 mm, 3 µm, Cat# 00F‐4453‐B0) column with an ultraviolet detector set at 214 nm.^[76, ¹⁰⁹, ^110] The peptides were resolved using a mobile phase consisting of 5% solvent A and 95% solvent B for 0.5 h at a velocity of 200 µL min⁻¹ (solvent A = H₂O, adjusted pH to 10.0 using NH₃·H₂O; solvent B = 80% ACN, adjusted pH to 10.0, using NH₃·H₂O).

OGP (4 µL) was incubated with or without 0.5 µL of rhCD10 (Cat# ab157051, Abcam) in a final volume of 200 µL of 50 × 10⁻³ m Tris‐HCl buffer (pH 7.5) for 1 h at 37 °C. To detect the cleavage sites of CD10 on OGP, enzymatic hydrolysis products of OGP after incubation with rhCD10 were used for HPLC‐MS/MS analysis.^[111] Briefly, the peptides were first loaded onto peptide trap columns (300 µm × 5 mm, 5 µm, Cat# 160 454, Thermo Fisher Scientific) and then separated on analytical columns (75 µm × 150 mm, 3 µm, Cat# 160 321, Thermo Fisher Scientific). Peptides bound to the columns were eluted with ACN in water (4–72% (v/v)) containing 0.1% FA at a flow rate of 300 nL min⁻¹ for 40 min. The MS analysis was performed online with an Orbitrap spectrometer (Thermo Scientific Q Exactive; Thermo Fisher Scientific). Full‐mass and MS/MS scans were acquired as previously described.^[112]Peak list files were analyzed against an in‐house constructed database based on the sequence of OGP using Mascot software. Peptides with at least four amino acids were included in the analysis. The FDR for the peptides was set to 0.05.