Supervisors info:
Νικόλαος Θωμαΐδης, Καθηγητής, Τμήμα Χημείας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
Allergy is an intense response of the immune system due to a type of antigen, the allergen. Allergens may be found in different foods and refer to proteins or peptides responsible for the allergenic activity. Most of these allergens are generally resistant to food processing. For example, peanut allergens are the most common cause of fatal anaphylaxis caused by food because, heat treatment of the food can increase their allergenicity and the reactions can be activated by some micrograms of the proteins. This research aims at the determination of allergenic proteins from nuts present in foods, such as chocolates, with ultra-High Performance Liquid Chromatography - tandem mass spectrometry (uHPLC-MS/MS). Until now, ELISA was been applied to allergen determination. LC-MS/MS technique is regarded as a new approach to detect multiple allergens. In this study an uHPLC-MS/MS method was developed and applied for the simultaneous identification and determination of the traces of tree nut allergens (peanut, pecan, pistachio, hazelnuts). Method’s protocol includes extraction of protein, trypsin digestion, purification with SPE and LC-MS/MS injection. Method’s parameters, such as time of digestion, were optimized. Moreover, LC-MS/MS detection was achieved based on subsequent peptides generated by enzymatic digestion. Specifically, for every peptide (two for every allergenic protein), the molecular mass and their two products were determined. Despite, time-consuming sample preparation, the developed method has many advantages. The method is characterized by high sensitivity, low limits of detection and it can provide simultaneous analysis of multiple tree nut allergens in a single preparation and a single injection step.
Keywords:
food allergens, trypsin digestion, peanut, tree nut, LC-MS/MS
