Disulfide proteome analysis of buckwheat seeds to screen putative allergens

Accumulating evidence suggests a correlation between disulfide bonding and the allergenicity of proteins. Also, a significant characteristic of most food allergens is that they are stable to proteases. We sought to identify putative allergens of buckwheat seed comprehensively by combining a disulfide proteome technique with an in vitro digestibility test. First, a dilute acetic acid fraction of buckwheat seed was found to be rich in disulfide proteins. Internal amino acid sequence analyses of these proteins showed that most of them were known allergens or putative allergens sharing high amino acid sequence similarities to known allergens. Next, the fraction was subjected to in vitro protease digestion, which revealed relatively large fragments that were resistant to prolonged enzymatic digestion. These protease-resistant fragments contained disulfide bonds that should have protected the potential enzyme cleavage sites by forming compact structures. These results confirm and extend our knowledge of the correlations among the disulfide bonding of proteins, their protease stability, and their allergenicity. Also, these observations suggest a new strategy to identify putative allergens by proteomic approaches as well as to mitigate them.