Efficient Protein Digestion Protocol

Proteins were digested by trypsin and LysC (Promega). trypsin hydrolyzes the ester bonds on the carboxyl side of Arg, while trypsin and LysC hydrolyze the ester bonds on the carboxyl side of Lys. The combination of trypsin and LysC enhances the efficiency of digestion. Therefore, trypsin/LysC digestion was carried out as described previously [27 (link)]. To the supernatant obtained from the above steps, 90 μL of 6 M urea was added and shaken for about 10 min at RT using a tube mixer. Then, 360 μL of 0.1 M Tris-HCl (pH 8.5) was added, and ultrasonic treatment and standing on ice were repeated twice for 30 s using an ultrasonic washer (Branson 2510, Danbury, CT, USA) to resuspend the protein precipitate. Next, 10 μL of 0.5 mg mL⁻¹ LysC solution and 25 μL of 1% Protease Max solution (Promega, Nacka, Sweden) were added and mixed by tapping, and the samples were incubated at 25 °C for 3 h. Finally, 10 μL of 0.5 mg mL⁻¹ trypsin solution was added and mixed by tapping, and the samples were incubated at 37 °C for 16 h.