Mass Spectrometric and Peptide Chip Epitope Analysis on the RA33 Autoantigen with Sera from Rheumatoid Arthritis Patients

As the potential of epitope chips for routine application in diagnostics relies on the careful selection of peptides, reliable epitope mapping results are of utmost interest to the medical community. Mass spectrometric epitope mapping, in combination with peptide chip analysis, showed that autoantibodies from patients who suffered from rheumatoid arthritis (RA) were directed against distinct surface structures on the full-length human autoantigen RA33 as well as against partial sequences. Using the combined mass spectrometric epitope extraction and peptide chip analysis approach, four sequence motifs on RA33 emerged as immuno-positive, showing that epitopes were not randomly distributed on the entire RA33 amino acid sequence. A sequential epitope motif (²⁴⁵GYGGG²⁴⁹) was determined on the C-terminal part of RA33 which matched with the Western blot patient screening results using the full-length protein and, thus, was regarded as a disease-associated epitope. Other epitope motifs were found on N-terminal partial sequences (⁵⁹RSRGFGF⁶⁵, ¹¹¹KKLFVG¹¹⁶) and again on the C-terminal part (²⁶⁶NQQPSNYG²⁷³) of RA33. As recognition of these latter three motifs was also recorded by peptide chip analysis using control samples which were negative in the Western blot screening, these latter motifs were regarded as “cryptic epitopes”. Knowledge of disease-associated epitopes is crucial for improving the design of a customized epitope peptide chip for RA and mass spectrometric epitope mapping pivotally assisted with selecting the most informative peptide(s) to be used for future diagnostic purposes.