Identification of (citrullinated) FN in RA synovial fluid. A. RA synovial fluid samples were depleted of albumin and separated by SDS-PAGE and stained with colloidal Coomassie Brilliant Blue. Subsequently, 18 equal slices were excised from the stained gel for both samples. The polypeptides present in the slices were digested with trypsin and analyzed by LC-MS/MS. The presence of citrullinated proteins in the gel was visualized by Western blotting using anti-modified citrulline (AMC) antibodies after modification of the proteins on the blot. The positions of molecular mass markers are indicated on the left (kDa). B. The positions of FN peptides detected in RA SF with LC-MS/MS for each of the 18 gel slices of both patient samples are schematically aligned with isoform 1 of fibronectin (FN1). The white bars represent peptides detected in RA1, the black bars peptides detected in RA2 and the grey bars peptides detected in both patients. The positions of the citrullinated residues detected are marked with asterisks. C. Schematic overview of the 15 different FN isoforms, resulting from alternative splicing events, documented in the UniProtKB database. The positions of the main alternatively spliced segments EDA, EDB and IIICS are indicated.