LC–MS/MS analysis of marker peptides for the detection of different mustard species and rapeseed in foods

Mustard is one of the allergens that must be declared on food labels, and accidental contamination of cereal-based products with mustard species has led to multiple food safety notifications in recent years. Due to the widespread use of Brassicaceae crops in agriculture and the high protein sequence homology among related species, selective identification of mustard contaminants remains analytically challenging. We hypothesized that species-specific peptide markers could be identified and used to discriminate closely related Brassicaceae species in durum wheat matrices. Proteins from white mustard (Sinapis alba), Indian mustard (Brassica juncea), black mustard (Brassica nigra), wild mustard (Sinapis arvensis), and rapeseed (Brassica napus) were extracted, digested with trypsin and chymotrypsin, and analyzed by LC–MS/MS. Candidate marker peptides were identified by full-scan analysis and evaluated by selected reaction monitoring (SRM) in durum wheat samples containing 1% contamination of individual species or a Brassicaceae mixture. Confirmed markers were further characterized by high-resolution mass spectrometry and sequence analysis. Species-specific peptide markers were identified for white mustard, Indian/black mustard, wild mustard, and rapeseed. Four markers were confirmed for white mustard, two for rapeseed, four for Indian/black mustard, and two for wild mustard, enabling reliable detection in wheat at 1% contamination. The proposed workflow enabled the differentiation of four analytical groups, namely Sinapis alba, Sinapis arvensis, Brassica napus, and a combined Brassica juncea/Brassica nigra group. The proposed method represents a complementary and confirmatory approach to existing PCR- and ELISA-based assays and may support future allergen-monitoring activities following broader analytical validation.