Multi-Allergen Detection: Determining Unique Nut and Tree Nut Peptide Markers Using Accurate-Mass Q-TOF LC/MS

The presence of hidden nut and tree nut allergens in foods can result in serious health issues, necessitating a method capable of detecting and quantifying them at trace levels using a single robust assay. To find specific peptide markers that can be used to determine the presence or absence of specific nuts in food, proteins unique to 11 tree nuts (almond, pecan, cashew, walnut, hazelnut, pine nut, brazil nut, macadamia nut, pine nut, chestnut, and coconut) and peanut were enzymatically digested and analyzed using accurate-mass Q-TOF LC/MS. Each marker peptide was selected by establishing its presence in raw and roasted nuts, processed and unprocessed food, abundance (sensitivity), sequence size, and uniqueness to a specific nut. The National Center for Biotechnology Information (NCBI) nr database searches were performed to confirm peptide identities, and to ensure that the marker peptides chosen were not present in other nuts or common food ingredients such as barley, corn, rice, soy, and wheat. Two marker peptides were selected for each tree nut, and four were selected for peanuts. Analysis of peptide digests from grains such as barley, corn, rice, quinoa, soy, and wheat did not present interferences. The peptide markers were tested to determine if they could be used to screen common foods for the presence of the 11 tree nuts and peanut at sub-ppm levels. Foods containing nuts as listed on the label showed a response for the correct nut. Foods processed using equipment also used to process other tree nuts or peanuts were in certain cases found to contain these other nuts.