2.3.2. Mass Spectrometry

After purification, acetonitrile was evaporated with the use of a rotary evaporator and the peptides were recovered by freeze-drying. Subsequently, the peptides were analysed on a Waters Micromass LCT TOF spectrometer (Waters UK, Wilmslow, UK) using electrospray ionisation in the School of Chemistry Mass Spectrometry facility. Prior to mass analysis, the peptide samples were dissolved in water. After the purification of the crude peptides, a purity of ≥95% was achieved and the theoretical mass agreed with the calculated mass displayed in Table 2.


**Table 2.** Purity and mass analysis of peptides.

2.3.3. Minimum Inhibitory Concentration (MIC) Values of Peptides

The minimum inhibition concentration (MIC) is used to determine the lowest concentration of antimicrobial agent needed to inhibit the visible growth of a bacteria strain after overnight incubation. All the bacteria strains were kindly provided by Dr Mark Webber of the Quadram Institute Bioscience (Norwich, UK; *Escherichia coli* (*E. coli*, I364), *Pseudomonas aeruginosa* (*P. aeruginosa*, PAO1) and *Staphylococcus aureus* (*S. aureus,* F77/NCTC8532)). Lysogen broth (LB broth) and agar were purchased from Sigma-Aldrich, Merck Life Science UK Limited, Dorset, UK. Fresh LB agar culture plates were prepared by pouring ≈10 mL of an autoclaved warm mixture of LB broth (2.5%) and bacteriological agar (1.5%) dissolved in dH2O. The agar plate was streaked and incubated overnight at 36 ◦C. A single bacterial colony was chosen and grown in 5 mL broth overnight under agitation at 36 ◦C. Subsequently, 50 μL of LB broth was added to wells 2–12 of a 96-well culture plate. AMPs were diluted to a concentration of 256 μg/mL, added to well 1, and diluted two-fold down to column 11. Column 12 was left empty, with no AMPs added. Then, 50 μL of the diluted overnight bacteria culture was added to the wells and incubated at 36 ◦C for 18 h to allow bacteria to grow. After incubation, the well plates were examined for bacterial growth and the lowest concentration of AMPs where clear liquid was observed was assumed to be the minimum inhibitory concentration. Three measurements for each peptide and against each type of bacteria were performed and the average values were obtained.
