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 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.

[1]  Anja Resemann,et al.  T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry. , 2003, Analytical chemistry.

[2]  D. Suckau,et al.  Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[3]  D. Suckau,et al.  Protein sequencing by ISD and PSD MALDI-TOF MS , 1998 .

[4]  David C. Lee,et al.  Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads , 2003, Journal of the American Society for Mass Spectrometry.

[5]  Martin C. Frith,et al.  Discovering Sequence Motifs with Arbitrary Insertions and Deletions , 2008, PLoS Comput. Biol..

[6]  Michael Karas,et al.  Matrix‐assisted laser desorption ionization mass spectrometry , 1991 .

[7]  K. Tomer,et al.  Epitope mapping by proteolysis of antigen-antibody complexes. , 2009, Methods in molecular biology.

[8]  S. Deininger,et al.  "Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping , 2004, Analytical and bioanalytical chemistry.

[9]  Yang Zhang,et al.  I-TASSER server for protein 3D structure prediction , 2008, BMC Bioinformatics.

[10]  Zbigniew Grzonka,et al.  Binding epitopes and interaction structure of the neuroprotective protease inhibitor cystatin C with beta-amyloid revealed by proteolytic excision mass spectrometry and molecular docking simulation. , 2009, Journal of medicinal chemistry.

[11]  M. Dougados,et al.  Diagnostic and predictive value of anti-cyclic citrullinated protein antibodies in rheumatoid arthritis: a systematic literature review , 2005, Annals of the rheumatic diseases.

[12]  J. Smolen,et al.  Neue Autoantikörper in der Diagnostik der rheumatoiden Arthritis , 2002, Zeitschrift für Rheumatologie.

[13]  Yang Zhang,et al.  Template‐based modeling and free modeling by I‐TASSER in CASP7 , 2007, Proteins.

[14]  G. Burmester,et al.  Autoantikörperprofile bei der rheumatoiden Arthritis , 2007, Zeitschrift für Rheumatologie.

[15]  L. Alfredsson,et al.  Immunity to citrullinated proteins in rheumatoid arthritis. , 2008, Annual review of immunology.

[16]  K. Tomer,et al.  Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis , 2008, Protein science : a publication of the Protein Society.

[17]  M. Przybylski,et al.  Epitope Extraction Technique Using a Proteolytic Magnetic Reactor Combined with Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry as a Tool for the Screening of Potential Vaccine Lead Peptides , 2005, European journal of mass spectrometry.

[18]  Eugen Damoc,et al.  Mass Spectrometric Approaches for Elucidation of Antigen—Antibody Recognition Structures in Molecular Immunology , 2007, European journal of mass spectrometry.

[19]  M. Glocker,et al.  Primary Structure Details of Haptoglobin α Chain Proteins from Human Plasma Samples are Resolved by Matrix-Assisted Laser Desorption/Ionization Quadrupole Ion Trap Time-of-Flight Multiple-Stage Tandem Mass Spectrometry Sequencing , 2004, European journal of mass spectrometry.

[20]  V. Neuhoff,et al.  Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250 , 1988, Electrophoresis.

[21]  M. Glocker,et al.  Proteome analysis reveals disease-associated marker proteins to differentiate RA patients from other inflammatory joint diseases with the potential to monitor anti-TNFalpha therapy. , 2004, Pathology, research and practice.

[22]  S. Gaskell,et al.  Epitope mapping of monoclonal antibodies by mass spectrometry: Identification of protein antigens in complex biological systems , 1998, Journal of the American Society for Mass Spectrometry.

[23]  P. James,et al.  Strategy for surveying the proteome using affinity proteomics and mass spectrometry , 2009, Proteomics.

[24]  B. Quilty,et al.  Prognostic criteria in rheumatoid arthritis: can we predict which patients will require specific anti-rheumatoid treatment? , 1997, Clinical and experimental rheumatology.

[25]  M. Glocker,et al.  Assignment of the inter- and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry. , 1993, Biochemistry.

[26]  M. Glocker,et al.  Differentiation of HELLP patients from healthy pregnant women by proteome analysis--on the way towards a clinical marker set. , 2006, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[27]  P. Lorenz,et al.  Probing the epitope signatures of IgG antibodies in human serum from patients with autoimmune disease. , 2009, Methods in molecular biology.

[28]  J. Tavaré,et al.  Methods in Molecular Biology: Protein kinases. , 1998 .

[29]  M. Takayama.  Matrix-assisted Laser Desorption/Ionization , 2004 .

[30]  A Tsugita,et al.  Sequence information of peptides and proteins with in‐source decay in matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry , 2000, Electrophoresis.

[31]  M. Glocker,et al.  MS characterization of apheresis samples from rheumatoid arthritis patients for the improvement of immunoadsorption therapy – a pilot study , 2009, Proteomics. Clinical applications.

[32]  M. Glocker,et al.  Rheumatoid Arthritis, a Complex Multifactorial Disease: On the Way Toward Individualized Medicine , 2006 .

[33]  C. Hager-Braun,et al.  The HIV-Neutralizing Monoclonal Antibody 4E10 Recognizes N-Terminal Sequences on the Native Antigen1 , 2006, The Journal of Immunology.

[34]  Yuki Nabeta,et al.  Human CD8 and CD4 T cell epitopes of epithelial cancer antigens , 2000, Cancer Chemotherapy and Pharmacology.

[35]  J. Leluk,et al.  A protein isolated from Escherichia coli, identified as GroEL, reacts with anti-beta spectrin antibodies. , 2003, Archives of biochemistry and biophysics.

[36]  M. Glocker,et al.  Matrix‐assisted laser desorption/ionization‐ quadrupole ion trap‐time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in‐gel tryptic digestion of haptoglobin derivatives from human plasma proteomes , 2003, Proteomics.

[37]  T. Köcher,et al.  Fragmentation of peptides in MALDI in-source decay mediated by hydrogen radicals. , 2005, Analytical chemistry.

[38]  Marina Cretich,et al.  Protein and peptide arrays: recent trends and new directions. , 2006, Biomolecular engineering.

[39]  J. Skolnick,et al.  Ab initio modeling of small proteins by iterative TASSER simulations , 2007, BMC Biology.

[40]  M. Przybylski,et al.  Mass spectrometric mapping of protein epitope structures of myocardial infarct markers myoglobin and troponin T. , 1996, Biochemistry.

[41]  W. Hassfeld,et al.  Clinical and immunological aspects of autoantibodies to RA33/hnRNP-A/B proteins — A link between RA, SLE and MCTD , 2004, Molecular Biology Reports.

[42]  Dieter Stoll,et al.  Proteome wide screening using peptide affinity capture , 2009, Proteomics.

[43]  R. Kominami,et al.  Anti-Sm autoantibodies cross-react with ribosomal protein S10: a common structural unit shared by the small nuclear RNP proteins and the ribosomal protein. , 1998, Arthritis and rheumatism.

[44]  J. Thornton,et al.  Continuous and discontinuous protein antigenic determinants , 1986, Nature.

[45]  G. Pruijn,et al.  Cross‐reactivity of the anti‐La monoclonal antibody SW5 with early endosome antigen 2 , 2002, Immunology.

[46]  Lawrence Steinman,et al.  Autoantibody profiling for the study and treatment of autoimmune disease , 2002, Arthritis research.