Università degli Studi della Tuscia, Department of Agriculture, Forests, Nature and Energy, Via S. Camillo de Lellis s.n.c. 01100, Viterbo, Italy.
J Proteomics. 2013 Mar 27;80:281-91. doi: 10.1016/j.jprot.2013.01.028. Epub 2013 Feb 9.
Wheat kernel albumins/globulins (A/G) and gluten proteins are responsible for baker's asthma and food allergy in atopic subjects. Although no commercial genetically modified wheats are currently being grown, they are under study and the allergenicity of GM products is a major concern. In order to establish the expected and unexpected effects of genetic transformation on allergenicity and also to carry out a safety assessment of genetic transformation, two GM wheat lines (bread and pasta wheat) transformed with endogenous genes were compared to their untransformed counterparts (wt), first by an allergenomic approach, and second, using ELISA with sera from patients suffering from food allergy to wheat and baker's asthma. The 2D immunoblots performed on sera from patients suffering from food allergy and baker's asthma on the A/G fraction of the four lines (two GM and two wt) revealed comparable IgE-binding profiles. A total of 109 IgE-binding spots were analyzed by mass spectrometry, and most of the proteins identified had already been described as allergens or potential allergens. Only two IgE-binding proteins were specific to one GM line. The concentration of specific IgE against the A/G fractions of GM wheat lines and their wt genotypes differed for some sera.
The originality of our paper is to relate the transformation of wheat lines with their potential allergenicity using patient sera, such focus has never been done before in wheat and should be of interest to the researches working in this field. Another interesting point of this paper is the study of two types of allergies (respiratory and food) on two wheat genotypes and their GM which reveals that some allergens already known in respiratory allergy could be involved in children suffering from wheat food allergy. In this paper we used a classical 2D proteomic analysis and the protein identifications were performed by mass spectrometry after spot picking and in gel trypsin hydrolysis. Concerning the LC-MS/MS analyses classical software and parameters were used as described in Material and methods. We worked on wheat which is actually not fully sequenced that was a difficulty; we therefore searched against two databanks (proteins and ESTs) in order to compare the results. Moreover all proteins reported in our paper were identified with at least three unique peptides. The identified proteins were checked for their potential allergenicity. In order to have a best interpretation of protein identified in terms of potential allergens, BLAST alignments were performed by using an allergen databank (SDAP). This allows the determination of the cross-reactivity of these identified proteins with known allergens of other species and also the prediction of a potential allergenicity.
麦醇溶蛋白/球蛋白（A/G）和谷蛋白是变应性患者中引起面包师哮喘和食物过敏的原因。尽管目前没有商业化的转基因小麦正在种植，但它们正在研究中，转基因产品的致敏性是一个主要关注点。为了确定遗传转化对致敏性的预期和意外影响，并对遗传转化进行安全性评估，我们将两种内源基因转化的转基因小麦品系（面包和意大利面小麦）与未转化的对应品系（wt）进行了比较，首先是通过过敏原组学方法，其次是使用来自患有小麦食物过敏和面包师哮喘的患者的血清进行 ELISA 检测。对来自患有食物过敏和面包师哮喘的患者的血清在四条线（两条 GM 和两条 wt）的 A/G 部分进行的 2D 免疫印迹显示，IgE 结合图谱相似。通过质谱法分析了总共 109 个 IgE 结合斑点，鉴定的大多数蛋白质已被描述为过敏原或潜在过敏原。只有两种 IgE 结合蛋白是一种 GM 线特有的。一些血清中 GM 小麦品系及其 wt 基因型针对 A/G 部分的特异性 IgE 浓度有所不同。
本文的创新性在于使用患者血清将小麦品系的转化与其潜在致敏性联系起来，这在小麦中从未有过此类研究，应该会引起该领域研究人员的兴趣。本文的另一个有趣之处是研究两种类型的过敏（呼吸道和食物）在两种小麦基因型及其 GM 上，这表明一些已经在呼吸道过敏中发现的过敏原可能与患有小麦食物过敏的儿童有关。在本文中，我们使用了经典的 2D 蛋白质组学分析，并且通过斑点挑取和凝胶内胰蛋白酶水解后进行质谱分析来进行蛋白质鉴定。关于 LC-MS/MS 分析，我们使用了如材料和方法中所述的经典软件和参数。我们研究的小麦实际上没有完全测序，这是一个困难；因此，我们搜索了两个数据库（蛋白质和 EST）以进行比较。此外，我们报告的所有蛋白质都至少被三个独特的肽鉴定。鉴定的蛋白质被检查其潜在的致敏性。为了对蛋白质在潜在过敏原方面的鉴定进行最佳解释，通过使用过敏原数据库（SDAP）进行 BLAST 比对。这允许确定这些鉴定的蛋白质与其他物种的已知过敏原的交叉反应性，以及预测潜在的致敏性。
