Profiling of Citrullinated Peptides in RA

The custom-made microarray was performed as previously described (7 (link)). In brief, 16 amino acid–long peptides that covered all arginine residues of 1610 extracellular matrix proteins and RA-related proteins were synthesized in situ (Roche NimbleGen). For TNC, there were a total of 217 peptides, including both native and citrullinated variants of each peptide synthesized. Samples tested for reactivity against TNC-derived peptides included synovial and plasma ACPA pools containing CCP-reactive antibodies from 26 and 38 RA patients, respectively (33 (link), 34 (link)) Paired synovial fluid and serum samples from 1 RA patient taken at 2 time points 10 years apart were also screened. ACPA pools were run at a concentration of 15 μg/mL, whereas synovial fluid and plasma or serum samples were diluted 1/100 and tested for citrulline reactivity using a NimbleGen MS200 Scanner (Roche NimbleGen). A peptide signal intensity variation (spot size) of 25 pixels was used as a basis for calculating the median fluorescence intensities. The cutoff for positive signals was defined as 5× the fluorescence intensity of the 98th percentile of values for a set of monoclonal antibodies without citrulline reactivity. These were included in the same experiment and were tested at the same time.