## 2 Results
According to the data for  
Figure 3: LIFT-MALDI-TOF/TOF product ion spectra of fusaricidin A ([M + H]\({}^{+}\) = 883.74) (**A**,**B**) and the novel derivatives of this compound with molecular masses [M + H + 71]\({}^{+}\) = 954.63 (**C**,**D**) and [M + H + 16]\({}^{+}\) = 899.43 (**E**,**F**), respectively. (**A**,**C**,**E**) cover the complete product ion spectra exhibiting _fragments a_ and the parent ions. (**B**,**D**,**F**) present the expanded mass range from \(m/z\) = 600–720 visualizing _fragments b_. fragment b of \(m/z=628.3\) and 642.2 they can be attributed to fusaricidins A and B bearing a C16-guanidino-3-hydroxy-hexadecameric acid as fatty acid constituent. In Figure 4A-C the complete sequence of fusaricidin A and its +71- and +16-variants obtained from series of b\({}_{n}\)- and y\({}_{n}\)-fragment ions are demonstrated. Indeed, unsubstituted fusaricidin A (Figure 3A) and its +16-derivative (Figure 3C) show the same peptide sequence, while the +71-variant (Figure 3B) is substituted at its allo-threonine residue in position 4 by an alanine forming an ester bond with its free hydroxyl group. This feature is corroborated by the sequence of the corresponding _fragments b_ of fusaricidin A (Figure 5A) and its +71-derivative (Figure 5B). These results were substantiated by the product ion spectra in Supplementary Materials (Figure S3A,B) showing their upper part in expanded version. Here, the value of fragment b\({}_{4}\) (\(m/z=769.14\)) is 71 mass units higher than b\({}_{4}\) of the unmodified fusaricidin A, indicating substitution of allo-Thr in position 4 by an alanine. Correspondingly, b\({}_{5}\) (\(m/z=812.63\)) is shifted to \(m/z\) 883.66 in the case of the +71-variant with a molecular mass of \(m/z=954.6\). This result is compatible with previous data obtained for fusaricidins C and D [10]. Figure 4: Sequence of fusaricidin A (precursor ion [M + H]\({}^{+}=883.74\)) (**A**) and its derivatives modified at the hydroxyl group of D-allo-Thr in position 4 by alanine (precursor ion [M + H + 71]\({}^{+}=954.78\)) (**B**) and by an additional hydroxyl group in its GDPH fatty acid side chain (precursor ion [M + H + 16]\({}^{+}=899.43\)) (**C**) derived from their LIFT-TOF/TOF product ion spectra. Structural analysis was based on three sets of b\({}_{n}\)-fragment ions, obtained either in their intact form or after elimination of H\({}_{2}\)O (b\({}_{n}\) (-H\({}_{2}\)O)) as well as of both H\({}_{2}\)O and NH\({}_{3}\) (b\({}_{n}\) (-H\({}_{2}\)O, -NH\({}_{3}\))), respectively. They were corroborated by the corresponding series of y\({}_{n}\)-ions. In Figure 6 and Table 2 the polymyxin products of the two investigated _P. polymyxin_ strains are shown. DSM 32871 produces two isomers of polymyxin E (E1 and E2) with molecular masses \([\text{M}+\text{H}]^{+}=1155.7\) and \(1169.8\) (see Table 2), while M1 forms two variants of polymyxin P (P1 and P2) with masses of \([\text{M}+\text{H}]^{+}=1177.4\) and \(1191.4\) (Figure 6C and Table 2) [38,44]. For both strains the isomers differ by a C8 or a C9 fatty acid. The protonated forms of these polymyxin families together with their alkali adducts are summarized in Figure 6 and Table 2. Both polymyxins have a high affinity for potassium, as shown by the \([\text{M}-\text{H}+2\text{K}]^{+}\)-ions indicating binding of two potassium atoms. The structure of polymyxins E studied by LIFT-MALDI-TOF/TOF MS is presented in Figure 7. Product ion spectra were taken either starting from the fatty acid constituent or the first amino acid Dab (1) (Figure 7A) as well as from Dab (5) and Leu (6) (Figure 7B) modelling the polymyxin structure from series of \(\text{b}_{\text{n}}\)- and \(\text{y}_{\text{n}}\)-ions. The fatty acid residues show masses of \(m/z=126.1\) and \(140.1\), respectively. The obtained sequence of polymyxin E is in accordance with the data reported in the literature [38, 39, 40, 41, 42]. The structure of polymyxin P was presented in a previous paper [36,44].