Bianco Mariachiara, De Palma Domenico, Pagano Antonio, Losito Ilario, Cataldi Tommaso R I, Calvano Cosima D
Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy.
Interdepartmental Research Center SMART, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy.
Int J Mol Sci. 2025 Apr 20;26(8):3891. doi: 10.3390/ijms26083891.
In recent years, sustainable agricultural practices in wheat cultivation have garnered significant attention, particularly those focused on minimizing pesticide and herbicide usage to safeguard the environment. One effective approach is green manuring, which entails rotating wheat with crops such as soybean and mustard to harness their natural pesticidal and herbicidal properties. While this method presents clear environmental advantages, it also poses a risk of cross-contamination, as these globally recognized allergens may unintentionally pass through wheat-based products. To protect consumers with allergies, there is an urgent need for a reliable analytical method to detect and quantify these allergenic proteins in wheat-derived foodstuffs. In this study, we assessed various protein extraction protocols to optimize the recovery of soybean and mustard allergens from wheat flour. The extracted proteins were analyzed using a bottom-up proteomics approach involving trypsin digestion, coupled with reversed-phase liquid chromatography and mass spectrometry in multiple reaction monitoring (MRM) mode. Two key allergenic proteins, Glycinin G1 and 11S Globulin, were selected as representative for soybean and mustard, respectively. The identified quantifier marker of Glycinin G1 was VLIVPQNFVVAAR ( 713.431), while FYLAGNQEQEFLK ( 793.896) and VFDGELQEGR ( 575.280) were designated as qualifier markers. The selection of specific marker peptides for mustard proved challenging due to the high structural similarity among proteins from and other members of the family. For 11S Globulin, FNTLETTLTR ( 598.319) was recognized as the quantifier marker, with VTSVNSYTLPILQYIR ( 934.019) serving as the qualifier marker. The developed method underwent thorough validation for linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, repeatability, and reproducibility, as well as potential matrix and processing effects. This strategy successfully facilitated the identification and quantification of soybean and mustard allergenic proteins in complex, processed food matrices, including naturally contaminated flour and cookies. These findings enhance food safety monitoring and regulatory compliance, thereby helping to mitigate allergen-related risks in wheat-based products.
近年来，小麦种植中的可持续农业实践受到了广泛关注，尤其是那些旨在减少农药和除草剂使用以保护环境的做法。一种有效的方法是绿肥种植，即将小麦与大豆和芥菜等作物轮作，利用它们的天然杀虫和除草特性。虽然这种方法具有明显的环境优势，但也存在交叉污染的风险，因为这些全球公认的过敏原可能会无意中进入小麦制品。为了保护过敏消费者，迫切需要一种可靠的分析方法来检测和定量小麦衍生食品中的这些致敏蛋白。在本研究中，我们评估了各种蛋白质提取方案，以优化从小麦粉中回收大豆和芥菜过敏原的效果。提取的蛋白质采用自下而上的蛋白质组学方法进行分析，包括胰蛋白酶消化，再结合反相液相色谱和多反应监测（MRM）模式的质谱分析。分别选择两种关键的致敏蛋白，大豆球蛋白G1和11S球蛋白，作为大豆和芥菜的代表。确定的大豆球蛋白G1定量标记物为VLIVPQNFVVAAR（713.431），而FYLAGNQEQEFLK（793.896）和VFDGELQEGR（575.280）被指定为定性标记物。由于与十字花科其他成员的蛋白质结构高度相似，为芥菜选择特定的标记肽具有挑战性。对于11S球蛋白，FNTLETTLTR（598.319）被识别为定量标记物，VTSVNSYTLPILQYIR（934.019）作为定性标记物。所开发的方法针对线性、检测限（LOD）、定量限（LOQ）、回收率、重复性和再现性以及潜在的基质和加工效应进行了全面验证。该策略成功地促进了在复杂的加工食品基质中，包括天然污染的面粉和饼干中，对大豆和芥菜致敏蛋白的鉴定和定量。这些发现加强了食品安全监测和监管合规性，从而有助于降低小麦制品中与过敏原相关的风险。
