For further proof of ncAA incorporation into nisin, HPLC-purified nisin(BocK) samples were analyzed by MALDI-TOF mass spectrometry. These measurements revealed that ncAA-modified nisin variants were synthesized at a very low level with the correct molecular weight (Table 1). Surprisingly, always a mixed population of full-size nisin(BocK) samples was determined: a larger one without His-tag and a smaller one with His-tag was found (data not shown). In the chosen genetic setup, the nisA reading frame starts with the ATG start codon of the N-terminal His-tag and contains a second, internal ATG codon originating from the first methionine of the leader, so two peptide variants (resulting from two different translation initiation sites) are possible. Selection of the untagged variant by the chosen purification method, differing stabilities of His-tagged and untagged variant or different folding of the peptide variants during biosynthesis in L. lactis are possible explanations for this phenomenon. Additionally, for nisin(I4BocK) and nisin(K12BocK), peaks corresponding to different dehydration states of the peptide were identified. Still, the main detected dehydration state corresponds to a nisin molecule with two or three rings, which explains the observed antimicrobial activity (Rink et al., 2007). As control, HPLC-purified peptide samples from cells grown without ncAA supplementation were utilized. As expected, MALDI-TOF analysis showed no peaks corresponding to full-size peptide products for this control (Figure 5).