Comparative Proteomic Analysis of Bacillus Cultures

To identify differentially enriched proteins in the supernatants of Btt and Bt407- cultures, we performed high resolution LC-MS/MS analysis by using an EASY-nLC 1200 (Thermo Fisher) coupled to an Exploris 480 mass spectrometer (Thermo Fisher). We separated peptides on 20 cm frit-less silica emitters (CoAnn Technologies, 0.75 µm inner diameter), packed in-house with reversed-phase ReproSil-Pur C₁₈ AQ 1.9 µm resin (Dr Maisch) and kept the column constantly at 50°C. We acquired the mass spectra in data-dependent acquisition mode as outlined in Sindlinger et al. [19 (link)]. We processed the raw data, by using the MaxQuant software version 2.0.3.0 [20 (link)]. MS/MS spectra were assigned to a custom Btt proteome assembly (Dr Heiko Liesegang, Institute of Microbiology and Genetics, University of Göttingen, unpublished) with default settings and match between runs; LFQ and iBAQ options were enabled. For further downstream analysis we used R [21 ]. After log₂ transformation, we imputed missing LFQ intensities based on quantile regression using imputeLCMD. To test for differential expression, we used LIMMA [22 (link),23 (link)]. Raw data were uploaded to the JPOST repository [24 (link)].