For MALDI–TOF–MS analysis, 1 μl of HPLC-purified sample was applied to the matrix target and treated as described earlier on a Voyager DE Pro MALDI-TOF spectrometer (Applied Biosystems) (van Heel et al., 2013). Data analysis was carried out with “Data Explorer” software version 4.0.0.0 (Applied Biosystems). Calculation of theoretical masses with ncAA-modified nisin variants was carried out with “massXpert”, version 3.4.0 (Rusconi, 2009).