Identification, in silico evaluation and in vitro assays of allergens from Sinapis arvensis (charlock mustard) and Brassica napus (rapeseed)

Currently, the allergenic proteins of some mustards, such as charlock mustard (Sinapis arvensis) and rapeseed (Brassica napus), have not been fully characterised at the protein level. The most widely used mustard varieties in food industry are white mustard (Sinapis alba) and Indian (Brassica juncea) mustard. Sinapis arvensis is a highly invasive weed that is not intentionally cultivated or used as a food ingredient. It can cause accidental contamination of harvested grain. Brassica napus, which is used in food and feed, has recently been approved as a Novel food (EU Regulation 2015/2283). The aim of this work was to identify the allergenic proteins of Sinapis arvensis and Brassica napus by in-gel tryptic digestion coupled with high-resolution mass spectrometry, and to compare them with the known allergens of Sinapis alba (Sin a 1 and Sin a 2) and Brassica juncea (Bra j 1). Allergenicity was assessed by in silico analysis and in vitro immunoblotting experiments using sera from individuals allergic to mustard and rapeseed. The major proteins identified were 11S globulins and 2S albumins. In addition, in silico analysis showed more than 80 % of sequence identity between the different mustard species, allowing for the comparison of known and previously uncharacterised allergenic proteins. These results were confirmed by in vitro immunoblotting tests. These findings highlighted the importance of a multiple approach strategy combining in silico, in vitro, and clinical data to assess the allergenic potential of alternative and novel proteins.