## 2 Results
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Strain DSM 32871: Polymyxin E** & **Strain M1: Polymyxin P** \\ \hline \multirow{2}{*}{\([\text{M}+\text{H},\text{Na},\text{K}]^{+}\)} & E1: 1155.7/1177.8/1193.8 & P1: 1177.4/1199.3/1215.4 \\  & E2: 1169.8/1191.8/1207.8 & P2: 1191.4/1213.4/1229.3 \\ \hline \multirow{2}{*}{\([\text{M}-\text{H}+2\text{K}]^{+}\)} & E1: 1231.9 & P1: 1253.9 \\  & E2: 1245.9 & P2: 1267.9 \\ \hline \multirow{2}{*}{\([\text{M}+\text{H}+146]^{+}\)} & E1: 1302.0 & P1: 1323.9 \\  & E2: 1316.0 & P2: 1337.9 \\ \hline \multirow{2}{*}{\([\text{M}+\text{H}+197]^{+}\)} & E1: 1352.9 & \multirow{2}{*}{
\begin{tabular}{c} \\ E2: 1366.9 \\ \end{tabular} } \\  & & E2: 1366.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Polymyxins produced by _P. polymyxin_ strains DSM 32871 and M1. Figure 5: Sequence of _fragments_\(b\) of fusaricidin A (**A**) and its derivative modified at the hydroxyl group of D-allo-Thr in position 4 by alanine (**B**) determined by LIFT-TOF/TOF mass spectrometry. Figure 6: MALDI-TOF mass spectra of the polymyxin families produced by _P. polymyxa_ DSM 32871 (**A**,**B**) and M1 (**C**) detected in the culture filtrates of these strains which were grown in the LOP medium for 48 h. Spectrum A was recorded in the mass range \(m/z=1180\)–1380 and shown in expanded form from \(m/z=1210\)–1380 in B.  
Interestingly, both for polymyxin E and P we observed a family of minor species apparent from Figure 6 at the higher mass range. For example, mass peaks were found at \(m/z=1302.0\) and \(1316.0\) for polymyxin E as well as at \(m/z=1323.9\) and \(1337.9\) for polymyxin P each showing a mass difference of approximately 146 Da relating to the protonated unsubstituted forms of E1/E2 and P1/P2, respectively (see Table 2). These species were sequenced by LIFT-MALDI-TOF/TOF MS. All of them showed the polymyxin sequence with a substitution of the Dab residue in position 4 by a threonine in combination with yet unknown structural elements at the C-terminal end of approx. 146 or 197 Da. This replacement was corroborated by nearest neighbor studies in Table 3 modelling the polymyxin structure by di-, tri-, tetra- and pentapeptide analysis. For example, in Figure 8 sequencing of the species with a molecular mass of \(m/z=1316.0\) is demonstrated which is a derivative of polymyxin E2 modified at its C-terminal end with a residue of approximately 146 Da. Similar results were achieved for the corresponding E1 derivative with a molecular mass of \(m/z=1302.0\). These compounds show a higher hydrophobicity than the original polymyxins demonstrated in Figures 6 and 7. In the HPLC-profile (Figure 2B) they were found in fractions 28 and 29 together with the tridecaptins, while the unsubstituted polymyxins E appeared in fraction 23. The nature of the additional elements in the sequence of the minor species have still to be elucidated. Figure 7: Sequence of the sodium adduct of polymyxin E2 (precursor ion [M + Na]* = 1191.8) derived from LIFT-MALDI-TOF/TOF product ion spectra which were taken either starting from the fatty acid constituent or the first amino acid Dab (1) (**A**) as well as from Dab (5) and Leu (6) (**B**) modelling the polymyxin structure from series of \(\mathrm{b_{n}}\)- and \(\mathrm{y_{n}}\)-ions.