Heating Differentiates Pecan Allergen Stability: Car i 4 Is More Heat Labile Than Car i 1 and Car i 2

Pecans are a staple in American cuisine and may be eaten raw but are often roasted or baked. Heating can alter pecan protein content and pecan allergen solubility. Three seed storage proteins (Car i 1, Car i 2, and Car i 4) commonly act as allergens and are recognized by IgE from pecan allergic individuals. Time resolved changes in the solubility of pecan allergens in response to heat were assessed by SDS-PAGE, immunoblot, and mass-spectrometry. Whole pecans from three different commercial sources were roasted for up to 24 min in an oven at 300◦F. Relatively smaller proteins such as Car i 1 remained soluble even after 24 min of heating and were stably observed by SDS-PAGE, immunoblot, and mass-spectrometry. However, the solubility of higher molecular mass proteins such as Car i 2 and Car i 4 decreased after 20 and 24 min of heating as reflected in SDS-PAGE and decreased antibody binding on immunoblot. Nonetheless, mass-spectrometric peptide characterization indicated that Car i 2 peptides remained relatively stable throughout heating. In contrast, Car i 4 was relatively more sensitive to heating and produced relatively fewer heating-insensitive peptides. A set of heat-resistant peptides for the reliable detection of three pecan allergens, Car i 1, Car i 2, and Car i 4, were identified.