Proteomic Profiling of Major Peanut Allergens and Their Post-Translational Modifications Affected by Roasting

Abstract

Post-translational modifications (PTMs) are covalent changes occurring on amino acid side chains of proteins and yet are neglected structural and functional aspects of protein architecture. The objective was to detect differences in PTM profiles that take place after roasting using open PTM search. We conducted a bottom-up proteomic study to investigate the impact of peanut roasting on readily soluble allergens and their PTM profiles. Proteomic PTM profiling of certain modifications was confirmed by Western blotting with a series of PTM-specific antibodies. In addition to inducing protein aggregation and denaturation, roasting may facilitate change in their PTM pattern and relative profiling. We have shown that Ara h 1 is the most modified major allergen in both samples in terms of modification versatility and extent. The most frequent PTM was methionine oxidation, especially in roasted samples. PTMs uniquely found in roasted samples were hydroxylation (Trp), formylation (Arg/Lys), and oxidation or hydroxylation (Asn). Raw and roasted peanut extracts did not differ in the binding of IgE from the serum of peanut-sensitised individuals done by ELISA. This study provides a better understanding of how roasting impacts the PTM profile of major peanut allergens and provides a good foundation for further exploration of PTMs.

Keywords: PTM profiling; allergy; high resolution mass spectrometry (HRMS); peanut allergen profiling; post-translational modifications; roasting; shotgun proteomics; western blot.

Figure 1

Raw and roasted PBS-based peanut extract (PEs) protein profiles. (a) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of peanut extracts (100 μg per lane) resolved on 12% polyacrylamide gel in reducing conditions. The grey boxes denote gel pieces excised and analyzed by nLC-MS/MS. (b) SDS-PAGE profiles of insoluble and soluble fractions analyzed with the equal volumes of raw and roasted peanuts. SDS-PAGE of the non-defatted and defatted crude peanut paste was obtained in reducing conditions by Laemmli buffer, phosphate-buffered saline (PBS) extracts were prepared from non-defatted and defatted peanut flour, while pellet extract was obtained by pellet extraction with denaturing buffer. (c) 2D SDS-PAGE protein profiles of raw and roasted peanut extracts.

Figure 2

Selected sequence stretches mapped as immunodominant epitopes of Ara h 1 (accession #P43237) and Ara h 6 (accession #Q647G9), with supporting peptides and their confident PTMs shown on the top of the sequence (AScore > 50 and manually inspected MS2 spectra).

Figure 3

1D immunoblots of protein post-translational modifications (PTMs) of the raw and roasted peanut extracts, purified allergens, and representative examples of relative PTM profiling by tandem mass spectrometry and PEAKS PTM relative profiling tool. (a) Immunoblot with rabbit IgG antibodies specific to methionine sulfoxide (OxM), methyl-lysine (MeK), hydroxy-proline (HyP), pyroglutamate (pGlu), carbamyl-lysine (Carb), and acetyl-lysine (AcK). Proteins were resolved on 14% polyacrylamide gel in reducing conditions. Goat anti-rabbit IgG conjugated with alkaline phosphatase was used as the secondary antibody. (b) PTM-profiling tool that delivers relative saturation of site-specific PTMs by PEAKS X Pro Studio. Legend: WB—western blot, Ra—raw PE, Ro—roasted PE, h1—Ara h 1, h2—Ara h 2, h6—Ara h 6, Ctrl—control without primary antibody.

Figure 4

IgE binding from the pool of patient sera to peanut extracts (PEs) in ELISA. (a) Inhibition of peanut-allergic patient IgE binding to raw PE (base-coat) by raw PE (turquoise line) and roasted PE (purple line). (b) Inhibition of peanut-allergic patient IgE binding to roasted PE (base-coat) by raw PE (turquoise line) and roasted PE (purple line).