2.4 Reproducibility assessment
A reproducibility assay was carried out with the 35 P. aeruginosastrains characterized by WGS. Each isolate was spotted in quadruplicate
(technical replicates) to assess the inter-spot or the technical
reproducibility of the method. Besides, two spectra were acquired per
spot to make an average spot spectrum. The biological reproducibility
was analyzed by subculturing overnight under the same conditions as
indicated above and acquiring protein spectra again in quadruplicates
(Candela et al., 2022;
Oberle et al., 2016). Data analysis was
carried out with Clover MS Data Analysis Software. The pipeline
described above was applied to the acquired spectra, which were
subsequently aligned to obtain an average spectrum per spot (shift
medium; linear mass tolerance 200 ppm). The same procedure was applied
to obtain a single average spectrum per day for each isolate. This
approach allowed the detection of common peaks present in all protein
spectra. The coefficient of variation (% CV) of the peak intensities
registered for each common peak after a 0.1 threshold applied was
calculated from TIC-normalized spectra.