Characterisation of genetically modified nisin molecules by Fourier transform ion cyclotron resonance mass spectrometry

Modified nisin molecules, synthesised by strains of Lactococcus lactis with deliberately mutated nisA genes, have been characterised using Fourier tra nsform ion cyclotron resonance mass spectrometry and electrospray ionisatio n. The predicted substitutions in the three nisin variants synthesised were first confirmed by precise measurement of the molecular mass (precision +/ -0.1 Da). Analysis of the lower intensity peaks in the mass spectra showed the presence of some minor components, notably hydrated molecules, in the f irst two samples. The third sample contained a major hydrated component tha t could be isolated in pure form by high-performance liquid chromatography. The engineered nisin molecules were further characterised by tandem mass s pectrometry using the sustained off-resonance irradiation collisionally-act ivated decomposition technique. This yielded a number of sequence ions that were compared with those measured in a previous study of nisin A itself. T he location of each substituent was deduced from the observed mass shifts o f the sequence ions. This permitted definitive confirmation of the predicte d substitutions. Out of several possible sites it was confirmed that positi on 33 contained the additional water molecule in the major hydrated form of one of the nisin variants.