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

Virginie Dutoit-Lefèvre,Patrick Vermersch,Vincent Sobanski,Lionel Prin,David Launay,Patricia Dussart,Philippe Chafey,Sylvain Dubucquoi,Sophie Duban-Deweer,Cédric Broussard,Didier Lefranc

doi:10.1371/journal.pone.0132142

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.

Highlights

Immunofluorescence, enzymatic and immunoprecipitation assays have been widely used for the identification of biomarkers in various diseases
While these issues have been resolved in proteomics by differential in-gel electrophoresis (2D-DIGE) [51], these steps remain a major limitation in serological proteomic analysis (SERPA) [52] even if different strategies have been proposed to circumvent them
The serological hybridizations (Fig 1A) showed heterogeneous patterns containing from 10 to 300 antigenic spots, whereas Coomassie colloidal blue (CCB) stained gel (Fig 1C) showed more than 1,000 protein spots distributed in the same ranges of pI and molecular weight (MW)

Summary

Introduction

Immunofluorescence, enzymatic and immunoprecipitation assays have been widely used for the identification of biomarkers in various diseases. Such redundant identification could be explained, at least in part, by the interpretation biases linked to the superimposition of images of 2-DE immunoblots and gels While these issues have been resolved in proteomics by differential in-gel electrophoresis (2D-DIGE) [51], these steps remain a major limitation in SERPA [52] even if different strategies have been proposed to circumvent them. These approaches are based either on the generation of a map of fixed benchmarks that offers additional visual anchors (so-called “landmark map” [4]) or the revelation of the proteomic map onto each immunoblot [34,36,53–55]. Even if they help final superimposition of the antigenic and the proteomic maps to target the protein spot to excise, these approaches still remain insufficient due to the persistence of an operator-dependent step to superimpose the different maps

Methods

Results

Conclusion