An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies

Abstract

Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.

Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome

“…Identification of bacterial antigens or autoantigens was often complicated due to different complexity levels in protein and immunopositive pattern or high antigenicity despite low protein abundance, interfering with post-experimental alignment [91,92,93]. In order to circumvent this, other attempts include the application of internal markers [94], additional staining steps [91], and alternative usage of protein arrays [95,96] or Luminex assays [97] based on recombinant proteins. 2DE partial immunoblotting as described here could complement screening approaches for linear protein antigens without bias to known antigens.…”

Immunoproteomics of Aspergillus for the development of biomarkers and immunotherapies

“…However, PTMs of proteins, which can also act as important parts of antigen epitopes, are mostly retained. Although the method has a high resolution in protein separation, solubility issues for hydrophobic proteins, sensitivity restrictions, and difficulties in allocating Western blot signals to protein spots can limit the identification of antigens [56]. Nevertheless, the SERPA approach is a powerful method that has been widely used to identify antigens in Aspergillus and other fungi as described in the next section.…”

Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases

“…Recently, a modified SERPA adapted from difference gel electrophoresis (DIGE) has been described as a fluorescence-based bidimensional immunoproteomics (FBIP) approach (7). The protein mixture is labeled with Cy3 fluorescent dye and loaded on a 2DE gel.…”