Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, Western blotting and Q-TOF mass spectrometry.

Peanut allergy is triggered by several proteins known as allergens. In this study, the complexity the peanut allergome is investigated with proteomic tools. The strength of this investigation resides in combining the high-resolving power and reproducibility of fluorescence two-dimensional differential gel electrophoresis with specific immunological detection as well as polypeptide sequencing by high-resolution mass spectrometry. Matching of the peanut proteins in 2D gels was achieved by differential labelling whereby peanut proteins and purified allergens (Ara h 1, Ara h 2 or Ara h 3/4) were run on the same gel. Ten protein spots on a mass line of ca. 63-68 kDa were likely to correspond to Ara h 1. Two doublets on two different mass lines at ca. 16 and 18 kDa matched with purified allergen Ara h 2. The basic and acidic sub-units of Ara h 3/4 were observed at masses of ca. 25 kDa and 40-45 kDa, respectively. Subsequently the antibody-binding capacity of spots corresponding to peanut allergens was investigated by Western blotting of 2D gels using antibodies (IgY) raised against Ara h 1, Ara h 2 and the recombinant 40 kDa sub-unit of Ara h 3/4. Final confirmation of the identity of the protein spots matched after 2D electrophoresis and identified by Western blotting was obtained by in-gel digestion of protein spots and analysis by quadrupole time-of-flight mass spectrometry. By using the method developed in our work, the location and identification of two different isoforms of the allergen Ara h 1, the allergen Ara h 2 and six isoforms of the allergen Ara h 3/4 in 2D peanut protein maps was established.