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United States Department of Agriculture

Agricultural Research Service

Research Project: REDUCING THE ALLERGENIC PROPERTIES OF PEANUTS

Location: Food Processing and Sensory Quality Research

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

Authors
item Chassaigne, Hubert - INST. FOR REF. MATERIALS
item Tregoat, Virginie - INST. FOR REF. MATERIALS
item Norgaard, Jorgen - INST. FOR REF. MATERIALS
item Maleki, Soheila
item Van Hengel, Arjon - INST. FOR REF. MATERIALS

Submitted to: Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 9, 2009
Publication Date: May 23, 2009
Citation: Chassaigne, H., Tregoat, V., Norgaard, J.V., Maleki, S.J., Van Hengel, A.J. 2009. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, western blotting and Q-TOF mass spectrometry. Proteomics. 72(3)511-526.

Interpretive Summary: Peanut allergy is triggered by several proteins known as allergens (Ara h 1, Ara h 2, or Ara h 3/4). These allergens were labeled with two different mass and charge, resolvable fluorescent dyes, and run on the same assay systems. Ten protein spots with a mass of ca. 63-68 kDa were likely to correspond to Ara h 1. Two doublets on two different mass 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. 20 kDa and 40-45 kDa, respectively. Subsequently, the antibody-binding capacity of spots corresponding to peanut allergens was using antibodies (IgY) raised in chicken 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 and identified with antibodies was obtained by in-gel digestion of protein spots and analysis of fragmented peptides spectrometry. The unambiguous presence of two different isoforms of the allergen Ara h 1, and six isoforms of the allergen Ara h 3/4 was tentatively established.

Technical Abstract: Peanut allergy is triggered by several proteins known as allergens. The matching resolution and identification of major peanut allergens in 2D protein maps, was accomplished by the use of fluorescence two-dimensional differential gel electrophoresis (2D DIGE), Western blotting and quadrupole time-of-flight (Q-TOF) mass spectrometry. Matching of the peanut proteins in 2D gels was achieved by differential labeling, whereby peanut proteins and purified allergens (Ara h 1, Ara h 2, or Ara h 3/4) were labeled 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 3/4 were observed at masses of ca. 20 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 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 Q-TOF mass spectrometry. The unambiguous presence of two different isoforms of the allergen Ara h 1, and six isoforms of the allergen Ara h 3/4 was tentatively established.

Last Modified: 7/22/2014