Peptide Variant Characterization by MALDI-TOF and Tricine-SDS-PAGE

After purification, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and tricine-SDS-PAGE (tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis) were employed to evaluate the production of peptide variants as previously reported (Liu et al., 2022 (link)). For MALDI-TOF analysis, 1-μl concentrated peptide sample was applied, using the same method as previously described (Liu et al., 2023 (link)). The amount of peptide used for tricine-SDS-PAGE analysis was purified from 20 mL of cell culture, i.e., 20-μl concentrated peptide sample. Specifically, the gels, consisting of a 16% separating gel and a 4% stacking gel, were prepared as previously reported (Schägger, 2006 (link)). For sample preparation, 5-μL loading buffer (10% SDS, 0.5% Bromophenol blue, 50% glycerol, 250 mM Tris-HCl, pH6.8) was mixed with the peptide sample, and the resulting mixture was heated at 50°C for 30 min. Additionally, a 5-μL portion of Unstained Low Range Protein Ladder (PageRuler, Thermo Fisher) was included as a protein marker and run alongside the peptide samples. After gel separation, the staining and destaining procedures were conducted following the previously reported method (Liu et al., 2022 (link)).