Within the complex wheat flour proteome, the gluten proteins have attracted most of the attention because of their importance in determining the functional properties of wheat flour doughs and their roles in human health conditions such as celiac disease and food allergies. However, certain non-gluten proteins also trigger immunological responses but may be present in flour in low amounts or obscured by the more abundant gluten proteins in two-dimensional gels of total protein preparations. Non-gluten proteins were preferentially extracted from the flour with a dilute salt solution and separated by two-dimensional gel electrophoresis. Proteins in 173 gel spots were identified by tandem mass spectrometry after cleavage with trypsin or chymotrypsin. Transgenic wheat lines in which specific groups of gluten proteins were suppressed by RNA interference were used to estimate the amount of carry-over of gluten proteins in the salt-soluble protein fraction. Fifty-seven different types of non-gluten proteins were identified, including 14 types that are known or suspected immunogenic proteins. The predominant proteins in 18 gel spots were gluten proteins. Some of these also contained non-gluten proteins. Analysis of the salt-soluble proteins from a transgenic line in which omega-1,2 gliadins were eliminated by RNA interference indicated that certain omega-1,2 gliadins were present in large amounts in the salt-soluble fraction and obscured relatively small amounts of beta-amylase and protein disulfide isomerase. In comparison, analysis of a transgenic line in which alpha gliadins were absent revealed that glyceraldehyde-3 phosphate dehydrogenase was a moderately abundant protein that co-migrated with several alpha gliadins. In this study, we constructed a proteomic map of the non-gluten protein fraction of wheat flour from the US wheat Butte 86 that complements a proteomic map of the total flour proteins developed previously for the same cultivar. Knowing the identities of low abundance proteins in the flour as well as proteins that are hidden by some of the major gluten proteins on two-dimensional gels is critical for studies aimed at assessing the immunogenic potential of wheat flour and determining which wheat proteins that should be targeted in future gene editing experiments to reduce the immunogenic potential of wheat flour.

中文翻译：

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破译小麦粉的免疫原性潜力：美国小麦 Butte 86 盐溶性蛋白质组的参考图谱。

在复杂的小麦粉蛋白质组中，面筋蛋白因其在确定小麦粉面团的功能特性及其在人类健康状况（例如乳糜泻和食物过敏）中的作用而受到关注。然而，某些非面筋蛋白也会引发免疫反应，但可能以少量存在于面粉中，或者被总蛋白制剂的二维凝胶中更丰富的面筋蛋白所掩盖。用稀盐溶液优先从面粉中提取非麸质蛋白质，并通过二维凝胶电泳分离。在用胰蛋白酶或糜蛋白酶切割后，通过串联质谱法鉴定了 173 个凝胶点中的蛋白质。使用 RNA 干扰抑制特定组面筋蛋白的转基因小麦品系来估计盐溶性蛋白质部分中面筋蛋白的残留量。鉴定了 57 种不同类型的非麸质蛋白，包括 14 种已知或疑似免疫原性蛋白。18 个凝胶点中的主要蛋白质是面筋蛋白。其中一些还含有非麸质蛋白质。对通过 RNA 干扰消除 omega-1,2 麦醇溶蛋白的转基因品系的盐溶蛋白的分析表明，某些 omega-1,2 麦醇溶蛋白大量存在于盐溶性部分中，并掩盖了相对少量的β-淀粉酶和蛋白质二硫键异构酶。相比下，对不含 α 麦醇溶蛋白的转基因品系的分析表明，甘油醛 3 磷酸脱氢酶是一种适度丰富的蛋白质，可与几种 α 麦醇溶蛋白共同迁移。在这项研究中，我们构建了来自美国小麦 Butte 86 的小麦粉的非麸质蛋白质部分的蛋白质组图，该图补充了之前为同一品种开发的总面粉蛋白质的蛋白质组图。了解面粉中低丰度蛋白质的特性以及二维凝胶上一些主要面筋蛋白所隐藏的蛋白质对于旨在评估小麦粉的免疫原性潜力和确定哪些小麦蛋白应该是至关重要的研究。未来基因编辑实验的目标是降低小麦粉的免疫原性潜力。