Mass spectrometric and peptide chip characterization of an assembled epitope: analysis of a polyclonal antibody model serum directed against the Sjøgren/systemic lupus erythematosus autoantigen TRIM21

Abstract

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.

Scalable high-density peptide arrays for comprehensive health monitoring

“…Peptide arrays have been used to assay kinase activity and inhibitors 23,31 , map monoclonal antibodies 32 , identify autoantigens 33 , even bind proteins 34 and cells 35 . The synthesis fidelity and peptide density of these arrays make it feasible to tile the entire human proteome or the proteomes of most human pathogens for studies of autoimmunity or vaccine development 23,26,36 .…”

A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures

“…Details on the sequence and production of recombinant human tripartite motif 21 (rhTRIM21) and recombinant human glucose-6-phosphate isomerase (rhGPI) protein have been described (Al-Majdoub et al, 2013a;Al-Majdoub et al, 2013b). Each protein (100 μg; 1 μg/μl, dissolved in 50 mM sodium phosphate, 1000 mM sodium chloride, and 8 M urea, pH 7.4) was reduced with 10 μl of freshly prepared 10 mM dithiothreitol in 100 mM ammonium bicarbonate solution and incubated at 56°C for 30 min, despite the fact that heating results in (partial) carbamylation of amino groups.…”

“…Disease-specific epitope peptides have been suggested to be used instead of full-length antigens to overcome current diagnostic limitations, and specialized arrays with peptides/epitopes of importance for a given disease (disease-specific arrays) are desired in the clinic for patient screening and/or disease diagnostics (Andresen and Grotzinger, 2009). Consequently, epitope mapping and detailed characterization of binding properties have become important for designing the so-called next generation chip arrays (Al-Majdoub et al, 2013a;Al-Majdoub et al, 2013b). To fulfill these tasks, fast and miniaturized isolation and preparation procedures are needed to gain access to intact antibody-epitope complexes.…”

High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients

“…Information about the precise epitopes is thus potentially relevant for MS diagnosis, prognosis, and monitoring (27). However, it should be noted that peptide microarrays are in general not suited to detect conformational epitopes, even if secondary structure conformations such as an alpha-helix can be formed by some short peptides (28). Different research groups have already applied microarrays to profile the autoantibody repertoire of MS patients against selected sets of peptides.…”