Peptide stability test.

Proteolytic digestion of CLP-4 was performed using tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK) trypsin (Thermo Fisher Scientific, Grand Island, NY) or chymotrypsin (ProteoChem, Hurricane, UT) immobilized on beaded agarose, according to manufacturer's recommendations. Proteases immobilized on beaded agarose helped eliminate enzyme digest contamination and thus simplify sample analysis. Briefly, 100 μl of resin containing 20 TAME (p-toluenesulfonyl-l-arginine methyl ester) units of immobilized TPCK trypsin or chymotrypsin was washed three times with 500 μl of digestion buffer (0.1 M NH₄HCO₃ [pH 8.0]); the gel was then suspended in 200 μl of digestion buffer, and 1 mg of CLP-4 was added for proteolytic digestion. After incubation at 37°C for 1, 2, 4, 18, and 24 h, 20 μl of treated samples was collected and analyzed by analytical RP-HPLC.