Two-dimensional electrophoresis 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 x 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.
Thebault
S
S
INSERM U519, Faculté Mixte de Médecine-Pharmacie, Hôpital Charles Nicolle, Rouen, France.
Gilbert
D
D
Machour
N
N
Marvin
L
L
Lange
C
C
Tron
F
F
Charlionet
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D015153:Blotting, Western / Q000379:methods; D002308:Cardiolipins / Q000276:immunology*; D002457:Cell Extracts; D003429:Cross Reactions; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000276:immunology; D018922:HL-60 Cells; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000276:immunology*; D000090243:Nucleophosmin; D010750:Phosphoproteins / Q000276:immunology*; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins / Q000276:immunology*; D012710:Serum Albumin, Bovine / Q000276:immunology; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014407:Tumor Cells, Cultured; D053482:beta 2-Glycoprotein I; D000099207:SS-A Antigen
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D002308:Cardiolipins; D002457:Cell Extracts; D006023:Glycoproteins; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D010750:Phosphoproteins; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D053482:beta 2-Glycoprotein I; D000099207:SS-A Antigen; C000716967:NPM1 protein, human; C000715570:Npm1 protein, mouse; C116006:RO60 protein, human; C439768:hnRNP A2