Mass spectrometric analysis of allergens in roasted walnuts

• Roasting has protein-specific effects on the quantification of walnut allergens.
• Sample preparation method and heat treatment level also affected quantification.
• Roasting can increase digestibility of some proteins and impact quantification.
• Potential Maillard adducts were identified on walnut proteins.

Thermal processing of allergenic foods can induce changes in the foods' constituent allergens, but the effects of heat treatment are poorly defined. Like other commonly allergenic tree nuts, walnuts often undergo heat treatment (e.g. roasting or baking) prior to consumption. This study evaluated the changes in solubility and detectability of allergens from roasted walnuts using tandem mass spectrometry methods. Walnuts were roasted (132 °C or 180 °C for 5, 10, or 20 min) and prepared for LC-MS/MS using sequential or simultaneous extraction and tryptic digestion protocols. The LC-MS/MS data analysis incorporated label-free quantification of relevant allergens and Maillard adduct screening. In some proteins (2S albumin, LTP, and the 7S globulin N-terminal region) minor changes in relative abundance were observed following roasting. The mature 7S and 11S globulins, however, showed significantly increased detection following roasting at 180 °C for 20 min when using the simultaneous extraction/digestion protocol, possibly due to increased digestibility of the proteins. The results of this study indicate that individual walnut allergens respond differently to thermal processing, and the detection of these proteins by LC-MS/MS is dependent on the protein in question, its susceptibility to proteolytic digestion, the degree of thermal processing, and the sample preparation methodology.SignificanceUnderstanding the behavior of food allergens in the context of relevant food matrices is critical for both food allergen management and for elucidating matrix and processing-associated factors influencing protein allergenicity. The use of mass spectrometry to identify food allergens and detect allergenic food residues has been increasingly developed due to the advantages associated with the direct, sequence-level analysis possible with MS. To date, however, few studies have implemented MS technology to analyze the effects of thermal processing on allergenic food proteins. The MS analysis results presented in this study revealed not only information about the molecular-level effects of roasting on walnut allergens but also data pertinent to the development of MS-based detection methods for walnut residues in food products.

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