Figure 3. Non-enzymatic cyclization of cytolysin S peptide in vitro.

a) ESI LC-MS analysis of the linear core peptide of CylL_S in its oxidized (disulfide) form. Extracted ion chromatogram (m/z = 1052) of the LC trace for oxidized CylL_S core peptide is shown in dark red and the corresponding mass spectrum in the insert. No peak corresponding to reduced CylL_S core peptide was detected in the LC trace. Oxidized CylL_S core peptide with a disulfide linkage, calculated [M+2H]²⁺: 1,051.53, monoisotopic mass; observed [M+2H]²⁺: 1,051.53, monoisotopic mass. b) ESI LC-MS analysis of oxidized and dehydrated core peptide of CylL_S. Extracted ion chromatogram (m/z = 1016.5) of the LC trace is shown with the corresponding mass spectrum inserted. Dhb1 was hydrolyzed to a ketone after leader peptide removal, resulting in a mass increase of 1 Da. Disulfide containing and dehydrated CylL_S core peptide (Dhb hydrolyzed), calculated [M–4H₂O–NH+O+2H]²⁺: 1,016.00, monoisotopic mass; observed [M–4H₂O–NH+O+2H]²⁺: 1,016.00, monoisotopic mass. c) ESI LC-MS analysis of non-enzymatically cyclized CylL_S core peptide. Extracted ion chromatogram (m/z = 1017) of the LC trace is shown. The mass spectrum corresponding to the major peak is shown in the insert. For fully cyclized cytolysin S, calculated [M–4H₂O+2H]²⁺: 1,016.52, monoisotopic mass; observed [M–4H₂O+2H]²⁺: 1,016.40, monoisotopic mass. Obu: 2-oxobutyrate, LP: leader peptide. Yellow line represents disulfide.