Simulated gastrointestinal digestion of Pru ar 3 apricot allergen: Assessment of allergen resistance and characterization of the peptides by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry Barbara Prandi 1 , Laura Farioli 2 , Tullia Tedeschi 1 , Elide Anna Pastorello 3 and Stefano Sforza 1 * 1 Department of Food Science, University of Parma, viale delle Scienze 17a, I-43124, Parma, Italy 2 Department of Laboratory Medicine, Hospital Niguarda Cà Granda, Piazza Ospedale Maggiore 3, 20162, Milano, Italy 3 Unit of Allergology and Immunology, Hospital Niguarda Cà Granda, Piazza Ospedale Maggiore 3, 20162, Milano, Italy RATIONALE: Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceæ family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. METHODS: Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH 4 HCO 3 at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. RESULTS: The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500–7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. CONCLUSIONS: An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology. Copyright © 2012 John Wiley & Sons, Ltd. Allergy is a hypersensitivity reaction initiated by immunolo- gical mechanisms and the antibody responsible for an allergic reaction typically belongs to the IgE isotype (IgE-mediated allergy). [1] A food allergy is an adverse immune response to a food protein. When some predisposed individuals eat a food allergen, they might present symptoms ranging from respiratory, gastrointestinal, cutaneous and cardiovascular, up to anaphylactic shock. [2] Allergy to Rosaceae fruits is often seen in association with birch pollinosis, [3] but severe cases exist in which Rosaceae allergy can be observed in patients without this cross- reaction. [4] In these cases, the major allergens belong to the family of non-specific lipid-transfer proteins (ns-LTPs). [5] Lipid-transfer proteins are small (molecular weight (MW) = 9–10 kDa) and basic (pI = 8.8–10.0) proteins that have been purified from various plant sources. [6] They have a tridimen- sional structure characterized by an a-helical compact domain comprised of four helices connected by eight cysteine residues, [7] with a tunnel-like hydrophobic cavity able to accommodate lipophilic ligands. [8] ns-LTPs are involved in plant defense mechanisms against phytopathogenic bacteria and fungi and possibly in the assembly of hydrophobic protective layers of surface polymers, such as cutin. [5] There is a high cross- reactivity among ns-LTPs from different plant species, [9] due to their sequence and resulting epitope similarity. [10] The resistance to proteolysis of ns-LTPs is well known: [11] apple, [12] cherry, [13] grape, [14] and peach [15] have already been subjected to in vitro digestion in order to assess their digestibility. Apricots are widely grown in Europe, and allergic reactions are becoming more common, especially oral allergy syndrome. [16] * Correspondence to: S. Sforza, Department of Food Science, University of Parma, viale delle Scienze 17a, I-43124 Parma, Italy. E-mail: stefano.sforza@unipr.it Copyright © 2012 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2012, 26, 2905–2912 Research Article Received: 19 June 2012 Revised: 21 September 2012 Accepted: 23 September 2012 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2012, 26, 2905–2912 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6416 2905