Two‐dimensional electrophresis and mass spectrometry identification of proteins bound by a murine monoclonal anti‐cardiolipin antibody: A powerful technique to characterize the cross‐reactivity of a single autoantibody

Antigenic cross‐reactivity, i.e., the capacity of a single antibody to react with apparently dissimilar structures, is a common characteristic of autoantibodies produced during systemic lupus erythematosus (SLE), an autoimmune disease developed by humans and certain strains of mice. Characterization of the extent of cross‐reactivity of SLE‐related autoantibodies may help identify the immunogenic stimulus, or stimuli, of autoantibody‐secreting B‐lymphocytes. Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was combined with mass spectrometry (MS) to identify cell proteins recognized by a single monoclonal autoantibody (mAb 4B7), derived from an (NZW × BXSB)F1 mouse and selected based on its capacity to react with cardiolipin, that binds to elements in the cytoplasm and nucleoli of HEp‐2 cells as assessed by indirect immunofluorescence assay. Proteins from HL‐60 extract were separated by 1‐D and 2‐D PAGE. Western blotting with mAb 4B7 after SDS‐PAGE revealed four bands, two intensely labeled at 35 and 32 kDa, and two weaker ones at 20 and 60 kDa; three spots were detected after 2‐D PAGE. After trypsin in‐gel digestion of the three protein spots, MS yielded representative matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) Reflector or quadrupole‐time of flight (Q‐TOF) spectra. The three corresponding proteins were identified as the nucleolar phosphoprotein B23 (nucleophosmin), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 60 kDa Ro/SS‐A RNP. Thus, these results showed that 2‐D PAGE combined with MS constitutes a sensitive and powerful technique to characterize the full extent of cross‐reactivity of a single mAb and may constitute a new approach to further characterize the immunogenic cellular components involved in the breakage of B‐cell tolerance observed in SLE.

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