Criterion xt gradient gels

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4–12% Criterion XT gradient gels (BioRad, Hercules, CA, USA) with XT MES running buffer (BioRad, Hercules, CA, USA). Gels were run at 125 V for 1 h along with a pre-stained protein standard (Thermo Fisher Scientific, Waltham, MA, USA) and stained for 1.5 h in a 1:1 (v/v) mixture of Coomassie Brilliant Blue R-250 and colloidal Coomassie Brilliant Blue G-250 (Thermo Fisher Scientific, Waltham, MA, USA). After washing in Millipore water overnight to remove excess dye, the stained gels were scanned and analyzed. From selected gel lanes, the protein bands were excised for tryptic digestion [38 (link)] and reconstituted in 50 μL of 1% formic acid in water. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) and subsequent data collection were performed as described previously [7 (link)].

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.