MALDI-TOF mass spectrometry.

For colony mass spectrometry (CMS), bacterial colonies of P9X and G10X mutants were collected with sterile plastic loops and mixed with 50 μl of 70% isopropyl alcohol (IPA) containing 0.1% trifluoroacetic acid (TFA). The suspension was vortexed, the cells were centrifuged in a benchtop centrifuge at 8,260 × g for 2 min, and the supernatant was removed for analysis. For matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry of nisin M cell-free supernatant (CFS) was purified as follows; a 1% inoculum of nisin mutant-producing strains was grown overnight in 50 ml clarified tryptone-yeast extract (TY) broth and incubated overnight at 30°C. Following incubation, cells were centrifuged at 5,000 rpm for 20 min at 4°C. CFS was removed and passed through a 1-g (6-ml) Strata C₁₈ E column (Phenomenex) pre-equilibrated with 6 ml methanol (Fisher Scientific, UK) and 6 ml HPLC-grade H₂O. The column was washed with 12 ml of 30% ethanol, and nisin was eluted using 5 ml of 70% isopropanol–0.1% TFA. Mass spectrometry in all cases was performed with an Axima TOF² MALDI-TOF mass spectrometer (Shimadzu Biotech, Manchester, UK). A 0.5-μl aliquot of matrix solution (α-cyano-4-hydroxycinnamic acid [CHCA], 10 mg ml⁻¹ in 50% acetonitrile–0.1% [vol/vol] TFA) was placed on the target and left for 1 to 2 min before being removed. The residual solution was then air dried, and the sample solution (resuspended lyophilized powder or CMS supernatant) was positioned on the precoated sample spot. Matrix solution (0.5 μl) was added to the sample and allowed to air dry. The sample was subsequently analyzed in a positive-ion linear mode.