Extracellular Bacterial Protein Profiling

Extracellular bacterial proteins contained in feather-supplemented media were concentrated using the Vivaspin™ ultrafiltration spin columns (GE Healthcare Life Sciences) according to the manufacturers’ recommendations. Protein mixtures were subsequently separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% Criterion XT gradient gels (BioRad, Hercules, CA, USA) with XT MES running buffer at 125 V for 1.5 h. The molecular weights (kDa) of the separated proteins were estimated using pre-stained markers and unstained high-mass-precision protein markers. For LC-MS/MS analysis, protein bands were excised from the gel as 10 molecular weight blocks per lane, followed by tryptic digestion as described by [15 (link)]. LC-MS sample processing, data acquisition, and data processing were carried out as previously described [16 (link)]. MS BLAST was then used to search a database derived from the in silico translation of the Bac18 genome. Search parameters specifying mass measurement accuracy, minimum number of product ion matches per peptide, minimum number of product ion matches per protein, minimum number of peptide matches, and maximum number of missed tryptic cleavage sites are detailed in the Supplementary Materials.