Mass Spectrometric and Peptide Chip Epitope Mapping of Rheumatoid Arthritis Autoantigen RA33

The protein termed RA33 was determined to be one major autoantigen in rheumatoid arthritis (RA) patients and antiRA33 auto antibodies were found to appear shortly after onset of RA. They are often detectable before a final diagnosis can be made in the clinic. The aim of our study is to characterise...

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Bibliographic Details
Published in:European journal of mass spectrometry (Chichester, England) England), 2009-01, Vol.15 (6), p.747-761
Main Authors: El-Kased, R.F., Koy, C., Deierling, T., Lorenz, P., Qian, Z., Li, Y., Thiesen, H.-J., Glocker, M.O.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Antibodies, Monoclonal
Arthritis, Rheumatoid
Autoantigens - chemistry
Autoantigens - immunology
Blotting, Western
Chromatography, High Pressure Liquid
Cyanogen Bromide
Electrophoresis, Polyacrylamide Gel
Epitope Mapping - instrumentation
Epitope Mapping - methods
Heterogeneous-Nuclear Ribonucleoprotein Group A-B - chemistry
Heterogeneous-Nuclear Ribonucleoprotein Group A-B - immunology
Humans
Molecular Sequence Data
Protein Array Analysis - methods
Protein Structure, Quaternary
Protein Structure, Tertiary
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
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Summary:The protein termed RA33 was determined to be one major autoantigen in rheumatoid arthritis (RA) patients and antiRA33 auto antibodies were found to appear shortly after onset of RA. They are often detectable before a final diagnosis can be made in the clinic. The aim of our study is to characterise the epitope of a monoclonal antiRA33 antibody on recombinant RA33 using mass spectrometric epitope mapping. Recombinant RA33 has been subjected to BrCN cleavage and fragments were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequent in-gel proteolytic digestion and mass spectrometric analysis determined the partial sequences in the protein bands. Western blotting of SDS-PAGE-separated protein fragments revealed immuno-positive, i.e. epitope-containing bands. BrCN-derived RA33 fragments were also separated by high-performance liquid chromatography (HPLC) and immuno-reactivity of peptides was measured by dot–blot analysis with the individual HPLC fractions after partial amino acid sequences were determined. The epitope region identified herewith was compared to data from peptide chip analysis with 15-meric synthetic peptides attached to a glass surface. Results from all three analyses consistently showed that the epitope of the monoclonal antiRA33 antibody is located in the aa79–84 region on recombinant RA33; the epitope sequence is MAARPHSIDGRVVEP. Sequence comparisons of the 15 best scoring peptides from the peptide chip analysis revealed that the epitope can be separated into two adjacent binding parts. The N-terminal binding parts comprise the amino acid residues “DGR”, resembling the general physico–chemical properties “acidic/polar–small–basic”. The C-terminal binding parts contain the amino acid residues “VVE”, with the motif “hydrophobic–gap–acidic”. The matching epitope region that emerged from our analysis on both the full-length protein and the 15-meric surface bound peptides suggests that peptide chips are indeed suitable tools for screening patterns of autoantibodies in patients suffering from autoimmune diseases.
ISSN:1469-0667
1751-6838
DOI:10.1255/ejms.1040