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Peanuts are a major crop throughout the world, grown in more than 80 countries for use as a cheap source of protein and for oil. They are found in many types of foods but their presence is feared by those of us who suffer from peanut allergies and have to spend hours reading food labels. Ingesting just a trace can result in severe anaphylactic shock, which could be life threatening. Recent years have seen a steady rise in the reported numbers of people suffering from peanut allergies. So much so that about 0.6-0.8% of the US population and 0.5% of the UK population is estimated to be affected. Peanut sensitivity often manifests itself in children under five years old and the bad news is that up to 80% of them will carry the allergy into adulthood. However, there is also some good news. Several treatments involving immunotherapy are under development and a US professor from Duke University Medical Center has predicted that some form of immunotherapy should be available within 5 years. Other approaches to the problem involve the development of genetically modified allergen-free peanut plants or the removal of the allergens from nuts by processing, as announced in the USA in 2008. All of these developments raise two obvious questions. How can we know for certain that a peanut, or indeed a food, is peanut allergen-free? And how do we measure the allergen contents of peanuts? One research group in Germany has turned to proteomics for the answer because the allergens are all different kinds of protein. Ottmar Janssen from the University Hospital Schleswig-Holstein, Kiel, with co-researchers from the University of Kiel and the Research Center Borstel, compared the protein compositions of two varieties of peanut, paying particular attention to the allergens. The Indonesian Kacang Asin peanut, also known as the Balinese or Bali peanut, is reputed to contain relatively low levels of the major allergen Ara h 1, sometimes referred to as conarachin. Its composition was compared with that of the Virginia-type peanut which has more "normal" allergen levels. Since mere traces of allergens can cause strong interactions in humans, a sensitive, high-resolution method is required for testing. The team chose two-dimensional differential gel electrophoresis (2D DIGE) for comparison, backed up by mass spectrometry for identifying proteins of interest. This technique was deemed to be better than one-dimensional gel electrophoretic techniques which were unable to resolve the many closely related proteins and isoforms within a particular allergen family. The target proteins tend to exist in the nut as complexes with other proteins, so an alkaline extraction procedure, which dissociates the complexes and solubilises the proteins, was adapted. After differential labelling with the fluorescent CyDyes, the proteins from each variety were separated and analysed. More than 100 protein spots on the gels were identified by mass spectrometry. There was a great variation in the compositions of the two samples, with 11.8% of the Virginia peanut proteins and 18.9% of the Indonesian proteins more prominent than in the opposite variety. For instance, the full-length Ara h 1 was indeed absent from Indonesian samples, as reported, but it was present in Virginian. However, truncated forms of Ara h 1 were detected in Indonesian peanuts, both N-terminal and C-terminal in nature, one of which has been reported to display reactivity in immunoblots using patient sera. The levels of Ara h 2, the other protein regarded as a major peanut allergen, were reduced in the Indonesian samples and some fragments were also detected. These identifications and their relative levels were confirmed by Western blotting experiments using monoclonal antibodies and the sera of allergic patients. Further allergens and fragments corresponding to Ara h 3/4 and Ara 6 were also found in both varieties but at different levels. Overall, it is clear that 2D DIGE has the ability to differentiate between the protein/allergen content of peanuts and it can "detect minute amounts of [allergen] contaminants." It is expected to be useful for testing allergen-reduced varieties of peanut as well as allergen preparations prepared from different sources. Related links:
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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