Methods for prompt and accurate characterization of potential allergens are handy tools for the food industry. Buhler et al. (2015) describe a workflow for isolating, identifying and characterizing non-specific lipid transfer proteins (nsLTPs) in almonds (Prunus dulcis L.) that combines liquid chromatography-electrospray ionization-Orbitrap mass spectrometry (LC-ESI-MS) with in silico allergenicity testing1.
nsLTPs are highly conserved proteins belonging to the alpha amylase inhibitor, lipid transport and seed storage family found in the Rosaceae plant family. The physical structure of the LTPs contains elements that attract IgE, thus leading to an allergic response in sensitized individuals. These are common allergens causing sensitivities in the Mediterranean region. Pru p3, an LTP found in peaches, is well studied; structurally, it contains highly-conserved disulfide bridges, forming a molecular conformation that resists degradation by cooking or digestion. Thus, allergenicity is retained, meaning that the resulting allergic reaction is strong. Almonds in food must be declared, even if found at trace levels (European Union Regulation #1169/2011).
Since there has not been a full characterization of potential allergens in almonds, Buhler et al. looked at LC-ESI-MS for a rapid and efficient method to identify the LTPs. The researchers first ground almonds obtained from a local market before defatting and then extracting the proteins with hexane. They separated the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After staining the resulting gel for protein, the team concentrated on the molecular weight band within the 3-30kDa range. They used fractionation by ultrafiltration to separate the proteins in this range further. They then repeated the SDS-PAGE, excising the bands between 6.5 and 14.4kDa as the region of interest containing putative LTPs. Following in-gel trypsin digestion carried out overnight, the researchers analyzed the samples by LC-ESI-MS, with a UltiMate 3000 high performance liquid chromatography (HPLC) system coupled with an LTQ Orbitrap mass spectrometer (both Thermo Scientific).
On analysis of the spectral data generated, the researchers constructed the amino acid sequence for the almond LTP under investigation. They found that it comprised 92 amino acids, with a molecular weight of 9,579 Da. Further analysis following reduction treatment with tributylphosphine revealed a molecular weight shift that corresponded to the four disulfide bridges highly conserved among other LTPs.
The team examined the purified protein using UV analysis to confirm structural characteristics. They also ran the amino acid sequence and structural data through four web-based testing programs to confirm the novel almond LTP’s potential for allergenicity.
Buhler et al. conclude that high-resolution LTQ Orbitrap mass spectrometry is an accurate tool for characterizing the exact molecular weight of the purified almond protein and its peptide fragments. The resulting data aided identification of a novel isoform of nsLTP in almonds, with subsequent in silico testing suggesting its potential as an allergen.
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1. Buhler, S. et al. (2015) “Isolation and full characterisation of a potentially allergenic lipid transfer protein (LTP) in almond“, Food Additives & Contaminants: Part A, 32:5, 648-656, DOI: 10.1080/19440049.2015.1016123