Sorted cell pellets were lysed in 50 mM Tris-HCl pH 7.4, 5% SDS, and sonicated (Bioruptor Pico, Diagenode, USA). Protein concentration was measured using the BCA assay (Thermo Scientific, USA). From each sample, 20 μg of total protein was subjected to in-solution tryptic digestion using the suspension trapping (S-trap) method as previously described [49 (link)]. Liquid chromatography and Mass Spectrometry was performed as described previously [24 (link)], using split-less nano-Ultra Performance Liquid Chromatography, reversed-phase Symmetry C18 trapping column (Waters) and a T3 HSS nano-column (Waters) for desalting and separation, coupled to a quadrupole orbitrap mass spectrometer (Q Exactive HFX, Thermo Scientific). Data acquisition, processing and analysis was performed as described [20 (link)]. Differential protein abundance was tested using the glht R function by ANOVA followed by Tukey analysis on the intensity values using a logarithmic scale. Changes in protein abundance of at least 1.5 between conditions, with p.value < 0.05 were considered significant.
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