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 of 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 (2D DIGE) with specific immunological detection as well as polypeptide sequencing by high resolution mass spectrometry followed by sequence identification employing database searches. Matching of the peanut proteins in 2D gels was achieved by differential labelling whereby peanut proteins and purified allergens (Ara h1, Ara h 2 or Ara h ¾) were labelled with two different mass and charge-matched, spectrally resolvable fluorescent dyes and 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 ¾ were observed at masses of ca. 25kDa and 4045 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 h1, Ara h 2 and the recombinant 40 kDa sub-unit of Ara h ¾. Final confirmation of the identity of the protein spots matched after 2D DIGE analysis and identified by western blotting was obtained by in-gel digestion of protein spots and analysis of tryptic peptides by capillary LC and quadruple time-of-flight (Q-TOF) mass spectrometry. By using the method developed in our work, the location and identification of two different isoforms of the allergen Ara h1 and six isoforms of the allergen Ara h ¾ in 2D peanut protein maps was established.

CHASSAIGNE Hubert;  TREGOAT Virginie;  NORGAARD Jorgen;  MALEKI Soheila J.;  VAN HENGEL Adrianus; 

2009-04-08

ELSEVIER SCIENCE BV

JRC47865

1874-3919,   

http://dx.doi.org/10.1016/j.jprot.2009.02.002,    https://publications.jrc.ec.europa.eu/repository/handle/JRC47865,   

10.1016/j.jprot.2009.02.002,