SDS-PAGE and Proteomic Analysis

SDS-PAGE was performed with 4%–20% precast gels in Tris-based buffer system from SERVA Electrophoresis (#43289.01) according to the manufacturer’s instructions. PageBlue Protein Staining Solution (Fermentas, #R0571) was used for coomassie-staining the gels, images were taken with iPhone 6s (Apple) and the contrast was adjusted using ImageJ (version 1.52d). Global identification and quantification of MSU- and zymosan-binding proteins in the presence of different donor sera was done as previously described (25 (link)). In brief, eluted proteins were reduced with DTT, alkylated with acrylamide, and separated using SDS-PAGE (4%–20%, Sigma-Aldrich). Whole lanes were cut into three individual slices and proteins therein were in-gel digested with trypsin. Generated peptides were analyzed using an LC-MS system consisting of an Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 RSLC nanoflow system (Thermo Fisher Scientific). Raw data were analyzed with the Andromeda search engine implemented in MaxQuant software (version 1.5.3.30; www.maxquant.org). Proteins were identified based on a false discovery rate (FDR) of less than 0.01 on protein and peptide level.