We demonstrate the development of a mass spectrometry-based epitope-mapping procedure in combination with Western blot analysis that works also with antigens that are insoluble in nondenaturing buffers consuming minute amounts of antigen (approximately 200 pmol) and antibody (approximately 15 pmol), respectively. A polyclonal anti-TRIM21 rabbit antibody serum is applied as a model serum for future patient analyses to set up the system. The major epitope that is recognized by the anti-TRIM21 serum spans the central TRIM21 region LQ-ELEKDEREQLRILGE-KE, showing that immunization with a 139-amino acid residue long peptide resulted in a 'monospecific' polyclonal antibody repertoire. Protein structure investigations, secondary structure predictions, and surface area calculations revealed that the best matching partial sequence to fulfill all primary and secondary structure requirements was the four amino acid spanning motif 'L-E-Q-L', which is present in both the sequential and the α-helical peptide conformation. Peptide chip analyses confirmed the mass spectrometric results and showed that the peptide chip platform is an appropriate method for displaying secondary structure-relying epitope conformations. As the same secondary structures are present in vivo, patient antibody screening, e.g., to identify subgroups of patients according to distinct epitope antibody reactivities, is feasible.
Copyright © 2013 John Wiley & Sons, Ltd.