ABSTRACT
The hazelnut allergen Cor a 14 belongs to the 2S albumins, a family of heterodimeric seed storage proteins exhibiting a high degree of structural diversity. Given its relevance as an allergen and the potential to elicit severe reactions, elucidation of the sequence heterogeneity of naturally occurring Cor a 14 is essential for the development of reliable diagnostics and risk evaluation. We therefore performed a comprehensive survey on the proteoforms of Cor a 14 and determined their quantitative distribution in three different hazelnut cultivars by a combinatory HPLC-HRMS approach including bottom-up and intact mass analysis. Compared with the Cor a 14 prototype sequence, we identified three sequence polymorphisms, two of the small and one of the large subunit, and elucidated their specific pairing on the protein level. Furthermore, we located a pronounced microheterogeneity on the protein termini and, for the first time, provide data on varying proteoform patterns between different cultivars of an allergenic seed. Together, these data present the basis for a more detailed investigation on the allergenicity of Cor a 14 in different cultivars and constitute, to be best of our knowledge, the largest set of proteoforms so far reported for a 2S albumin.
Subject(s)
Allergens/chemistry , Antigens, Plant/chemistry , Polymorphism, Single Nucleotide , Protein Subunits/chemistry , Seed Storage Proteins/chemistry , Allergens/genetics , Allergens/isolation & purification , Amino Acid Sequence , Antigens, Plant/genetics , Antigens, Plant/isolation & purification , Chromatography, High Pressure Liquid , Corylus/chemistry , Gene Expression , Humans , Mass Spectrometry , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/isolation & purification , Protein Subunits/genetics , Protein Subunits/isolation & purification , Seed Storage Proteins/genetics , Seed Storage Proteins/isolation & purificationABSTRACT
The first combination of electrochemistry (EC), NACE, and ESI-MS to mimic the metabolic fate of drugs is described. While the combination of EC, HPLC, and ESI-MS has been used for this purpose before, NACE is able to deliver valuable additional information about possible metabolites of harmane when compared to HPLC. In this paper, NACE is used as a comprehensive separation technique in metabolism studies of harmane, a naturally occurring monoaminooxidase inhibitor, since it exhibits beneficial properties for the separation of polar compounds. Harmane is known to be metabolized via the oxidative metabolism catalyzed by cytochrome P450 enzymes, which are the most important metabolizing superfamily of enzymes in the human liver. The application of HPLC and NACE enabled the detection of 37 products in total, with 14 different mass-to-charge ratios. A total of 31 products could be detected in HPLC-MS and 26 in NACE-MS analysis. The combination of both NACE and RP-HPLC allows the identification of significantly more potential metabolites than any of the separation techniques alone.
