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.