Abstract RATIONALE: Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae 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.
|Titolo:||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|
PASTORELLO, ELIDE ANNA (Penultimo)
|Parole Chiave:||Adult ; Amino Acid Sequence ; Antigens, Plant ; Chromatography, High Pressure Liquid ; Chymotrypsin ; Female ; Food Hypersensitivity ; Gastric Acid ; Humans ; Hydrochloric Acid ; Hydrogen-Ion Concentration ; Immunoblotting ; Immunoglobulin E ; Male ; Middle Aged ; Molecular Sequence Data ; Peptide Fragments ; Protein Stability ; Prunus ; Sequence Alignment ; Spectrometry, Mass, Electrospray Ionization ; Trypsin ; Young Adult|
|Settore Scientifico Disciplinare:||Settore MED/09 - Medicina Interna|
|Data di pubblicazione:||30-dic-2012|
|Digital Object Identifier (DOI):||10.1002/rcm.6416|
|Appare nelle tipologie:||01 - Articolo su periodico|