Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry

After cation exchange chromatography, the partially purified BLIS was analyzed by one-dimensional SDS-PAGE (Mini-PROTEAN electrophoresis apparatus; Bio-Rad, Hercules, CA, USA) using a 4% (v/v) stacking gel and 15% (v/v) separating gel. A pre-stained molecular mass protein marker with a range of 7000–70,000 Da was used. The BLIS was separated at 180 V for 45 min, and the gel was divided into two identical vertical parts, one of which was stained and visualized using silver nitrate. The other part was used to identify antibacterial activity by first washing the gel three times with distilled water (40 min/wash) to remove excess SDS. Then, the gel was transferred to an MRS agar plate and overlaid with 10 mL of soft MRS agar containing the indicator microorganism (L. sakei LMG 2313) and incubated overnight at 30 °C. The molecular weight of the purified peptide after reverse-phase chromatography was further confirmed with matrix-assisted laser desorption ionization (MALDI)-TOF/MS (Voyager-DE RP; Applied Biosystems, Foster City, CA, USA), as described^34,35. Briefly, 0.5 μL of the sample was mixed with 0.5 μL of the matrix mixed with 15 mg/mL alpha-cyano-4-hydroxycinnamic acid and deposited on a ground steel MALDI plate (Bruker Daltonics, Billerica, MA, USA). The mass window was adjusted between 4000 and 10,000 Da in the linear positive ion mode, with an acceleration voltage of 25 kV. A peptide mass similarity search was performed using ExPASy with the TagIdent tool (http://www.pdg.cnb.uam.es/cursos/Leon_2003/pages/visualizacion/programas_manuales/spdbv_userguide/us.expasy.org/tools/tagident.html).